The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli

Madhusudhan, K. T.; Lorenz, D; Sokatch, John R.

doi:10.1128/jb.175.13.3934-3940.1993

Cited by 48 publications

(53 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The final product was biologically active in gel mobility shift assays. The sequence of the first 10 N-terminal amino acids of purified BkdR was Met Arg Lys Leu Asp Arg Thr Asp Ile Gly, which agreed with the predicted amino acid sequence (13).…”

Section: Resultssupporting

confidence: 61%

See 1 more Smart Citation

Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida

1995

Self Cite

View full text Add to dashboard Cite

The bkd operon of Pseudomonas putida consists of the structural genes encoding the components of the inducible branched-chain ketoacid dehydrogenase. BkdR, a positive regulator of the bkd operon and a homolog of Lrp of Escherichia coli is encoded by a structural gene adjacent to, and divergently transcribed from, the bkd operon of P. putida. BkdR was purified from E. coli containing bkdR cloned into pCYTEXP1, an expression vector. The molecular weight of BkdR obtained by gel filtration indicates that BkdR is a tetramer, and the abundance of BkdR in P. putida was estimated to be about 25 to 40 copies of the tetramer per cell. BkdR bound specifically to the region between bkdR and bkdA1, the latter being the first gene of the bkd operon. One BkdR-DNA complex was observed in gel mobility shift patterns. Approximately 100 bp was protected from the action of DNase I by BkdR, and the addition of L-branched-chain amino acids enhanced the appearance of hypersensitive sites in the protected region. There are four potential BkdR-DNA binding sequences in this region based on similarity to Lrp-binding consensus sequences. Like many other transcriptional activators, BkdR regulates expression of its structural gene. DNAs from several gram-negative bacteria hybridized to a probe containing bkdR, indicating the presence of bkdR-like genes in these organisms.Branched-chain ketoacid dehydrogenase of Pseudomonas putida is an inducible multienzyme complex produced by the organism grown in media with branched-chain amino or keto acids as carbon sources (15). The formation of branched-chain ketoacid dehydrogenase is repressible by glucose and NH 4 ϩ (27), and the effects seem to be additive. The components of branched-chain ketoacid dehydrogenase are E1 (the dehydrogenase-decarboxylase), E2 (the transacylase), and E3 (lipoamide dehydrogenase). The structural genes for these proteins are encoded by the bkd operon of P. putida, which has been cloned and whose nucleotide sequence has been determined (3-5). All four genes are tightly linked, and the operon is transcribed as a polycistronic message (5).An open reading frame upstream of, and divergently transcribed from, the bkd operon, encoding a protein, BkdR (13), with 37.5% amino acid sequence identity to Lrp, the leucineresponsive regulatory protein of Escherichia coli (33), was found. Lrp is a global transcriptional regulator in E. coli (6), the action of which can be antagonized or potentiated by leucine or may be insensitive to the presence of leucine. Expression of the ilvIH operon of E. coli is regulated by Lrp and is repressible by leucine. Lrp binds to several sites in the regulatory region of the ilvIH operon of E. coli (29) and causes DNA bending (30).Chromosomal mutations affecting bkdR resulted in failure to produce branched-chain ketoacid dehydrogenase, and mutations in bkdR were complemented in trans by both bkdR and lrp (13). These results suggest that BkdR acts as a positive transcriptional activator of the bkd operon. This article describes the cloning and overexpressi...

show abstract

Section: Resultssupporting

confidence: 61%

“…The deduced amino acid sequence of BkdR lacks tryptophan but contains 2.48 mol% tyrosine and 3.11 mol% phenylalanine (13). The molar extinction coefficient of BkdR was estimated to be 5,240 by the PeptideSort program of the Genetics Computer Group sequence analysis program (8).…”

Section: Resultsmentioning

confidence: 99%

Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida

1995

Self Cite

View full text Add to dashboard Cite

show abstract

“…Orf1 contains 164 amino acid residues, corresponding to a molecular mass of 18,690 Da. Orf1 shows 33% amino acid identity (52% similarity) to Lrp from E. coli and 35% identity (54% similarity) to BkdR, a transcriptional regulator from Pseudomonas putida (26). The alignment of all three proteins is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli

et al. 1995

View full text Add to dashboard Cite

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H ؉ / glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H ؉ -coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H ؉ /glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.

show abstract

“…The third family member, YbaO has no known effector. Effectors have been indentified for other members of the AsnC/Lrp family including L-leucine for Lrp (Willins, Ryan, Platko & Calvo, 1991), L-proline for PutR (Keuntje, Masepohl, & Klipp, 1995), L-glutamate for Grp (Peekhaus, Tolner, Poolman, & Kramer, 1995), L-methionine for MdeR (Inoue et al, 1997) and branched chain amino acids for BkdR (Madhusudhan, Lorenz, & Sokatch, 1993).…”

Section: Introductionmentioning

confidence: 99%

In-vitro Characterization of an L-Kynurenine-Responsive Transcription Regulator of the Oxidative Tryptophan Degradation Pathway in Burkholderia xenovorans

Hall¹,

Martí‐Arbona²,

Hennelly³

et al. 2013

JMBR

View full text Add to dashboard Cite

To study the transcriptional regulation of oxidative tryptophan degradation in Burkholderia, we used comparative genomics that focused on the operon containing the genes annotated as kynA, kynU and kynB. In all sequenced β-proteobacteria to-date, including Burkholderia, Ralstonia, Collimonas, and Cupriavidus species, there is a conserved AsnC/Lrp family transcriptional regulator (TR) gene located upstream and in the opposite strand of the operon encoding for the oxidative tryptophan degradation genes. In Burkholderia xenovorans the TR is Bxe_A0736. GST-Bxe_A0736 binds L-kynurenine with greater affinity and specificity than any other amino acid or tryptophan degradation product with a dissociation constant of ~82 ± 11 μM. DNaseI footprinting suggested that Bxe_A0736 protects a set of four degenerate, palindromic sequences within the intergenic region between Bxe_A0735 (kynB) and Bxe_A0736. The optimal consensus sequence obtained by analysis for these sites, ATATTCCGAATAT, closely resembles the sequence obtained with a protein binding microarray. Under our fluorescence anisotropy experimental conditions, 1 mM L-kynurenine increased the affinity of Bxe_A0736 for a portion of its promoter region. Our results are consistent with Bxe_A0736 acting as the TR that promotes the transcription of the oxidative tryptophan degradation genes in the presence of L-kynurenine while inhibiting the transcription of its own gene.

show abstract

The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli

Cited by 48 publications

References 36 publications

Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida

Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli

In-vitro Characterization of an L-Kynurenine-Responsive Transcription Regulator of the Oxidative Tryptophan Degradation Pathway in Burkholderia xenovorans

Contact Info

Product

Resources

About