Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

Govantes, Fernando; Molina-López, José; Santero, Eduardo

doi:10.1128/jb.178.23.6817-6823.1996

Cited by 53 publications

(34 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Total DNA from strain TFA was prepared as previously described (20). Southern blot analyses were performed using digoxigenin-dUTP-labeled probes and following the instructions of the manufacturer (Boehringer Mannheim).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

et al. 2004

View full text Add to dashboard Cite

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys 4 [2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NADbinding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.The organic solvent tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which share two carbon atoms. Tetralin is widely used as a degreasing agent and solvent for fats, resins, and waxes, as a substitute for turpentine in paints, lacquers, and shoe polishes, and also in the petrochemical industry in connection with coal liquefaction (19). A concentration of tetralin higher than 100 M inhibits bacterial growth (44). Its toxicity is partly due to its lipophilic character, which results in its accumulation in the cell membranes, thus leading to changes in their structure and function (46,47). In addition, tetralin also forms toxic hydroperoxides in the cell (17).A few bacterial strains which are able to aerobically grow on tetralin as the only carbon and energy source have been isolated (44). By the identification of accumulated intermediates, several reports suggest that some bacteria, such as Pseudomonas stutzeri AS39 (43), initially hydroxylate and further oxidize the alicyclic ring whereas others, such as Corynebacterium sp. strain C125 (45), initially dioxygenate the aromatic ring, thus indicating that aerobic metabolism of tetralin can be performed in different ways. Metabolism of tetralin has been best characterized in Sphingomonas macrogolitabida strain TFA. Biodegradation of tetralin by the strain TFA involves initial...

show abstract

Section: Methodsmentioning

confidence: 99%

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

et al. 2004

View full text Add to dashboard Cite

show abstract

“…For overexpression of wild-type and thnC mutants, E. coli NCM631/pIZ227 (Govantes et al, 1996) was transformed with the plasmids pIZ591, pIZ595, pIZ596, pIZ598, pIZ599, pIZ1100, pIZ1101, pIZ1102, pIZ1103, pIZ1104 or pIZ1105, bearing the different alleles of thnC fused to a His-tag. The resulting transformants were grown in LB liquid medium at 26˚C to OD 600 0?6-0?8.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting plasmids, together with pIZ591, were used to transform the overproducing strain NCM631/pIZ227 (Govantes et al, 1996). After induction of the transformed strains with IPTG, overproduction was very evident in all cases.…”

Section: Mutagenesis Of Thnc and Purification Of The Modified Gene Prmentioning

confidence: 99%

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

Andújar

Santero

2003

Microbiology

View full text Add to dashboard Cite

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increased k cat /K m at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increased k cat /K m using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal b-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5?5. In addition, Q198D and V175D mutants, which had altered K m , also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

show abstract

“…Both A. vinelandii and K. pneumoniae NifL are flavoproteins with flavin adenine dinucleotide as the prosthetic group (51,91,201). The NifL and NifA proteins form a complex in vivo and in vitro, and stoichiometric levels of the two proteins are required for effective modulation of NifA activity (82,90,162).…”

Section: Regulation Of Nitrogen Fixationmentioning

confidence: 99%

P_IISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

Arcondéguy

Jack

Merrick

2001

Microbiol Mol Biol Rev

398

456

View full text Add to dashboard Cite

SUMMARY The PII family of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world. First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, PII proteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism. More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control. The PII proteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism. There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell. In this review we discuss the history of the PII proteins, their structures and biochemistry, and their distribution and functions in prokaryotes. We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by PII proteins.

show abstract

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

Cited by 53 publications

References 39 publications

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

P_IISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

Contact Info

Product

Resources

About

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

Cited by 53 publications

References 39 publications

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

Contact Info

Product

Resources

About

P_IISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control