Dataset Information

Transcriptional activation of the bkd operon of Pseudomonas putida by BkdR.

ABSTRACT: Reinvestigation of the transcriptional start site of the bkd operon of Pseudomonas putida revealed that the transcriptional start site was located 86 nucleotides upstream of the translational start. There was a sigma 70 binding site 10 bp upstream of the transcriptional start site. The dissociation constants for BkdR, the transcriptional activator of the bkd operon, were 3.1 x 10(-7) M in the absence of L-valine and 8.9 x 10(-8) M in the presence of L-valine. Binding of BkdR to substrate DNA in the absence of L-valine imposed a bend angle of 92 degrees in the DNA. In the presence of L-valine, the angle was 76 degrees. BkdR did not bind to either of the two fragments of substrate DNA resulting from digestion with AgeI. Because AgeI attacks between three potential BkdR binding sites, this suggests that binding of BkdR is cooperative. P. putida JS110 and JS112, mutant strains which do not express any of the components of branched-chain keto acid dehydrogenase, were found to contain missense mutations in bkdR resulting in R40Q and T22I changes in the putative helix-turn-helix of BkdR. Addition of glucose to the medium repressed expression of lacZ from a chromosomal bkdR-lacZ fusion, suggesting that catabolite repression of the bkd operon was the result of reduced expression of bkdR. These data are used to present a model for the role of BkdR in transcriptional control of the bkd operon.

SUBMITTER: Madhusudhan KT

PROVIDER: S-EPMC178924 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Transcriptional activation of the bkd operon of Pseudomonas putida by BkdR.

Madhusudhan K T KT Hester K L KL Friend V V Sokatch J R JR

Journal of bacteriology 19970301 6

Reinvestigation of the transcriptional start site of the bkd operon of Pseudomonas putida revealed that the transcriptional start site was located 86 nucleotides upstream of the translational start. There was a sigma 70 binding site 10 bp upstream of the transcriptional start site. The dissociation constants for BkdR, the transcriptional activator of the bkd operon, were 3.1 x 10(-7) M in the absence of L-valine and 8.9 x 10(-8) M in the presence of L-valine. Binding of BkdR to substrate DNA in ...[more]

PMID: 9068646

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Project description:Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings.IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

Dataset Information

Transcriptional activation of the bkd operon of Pseudomonas putida by BkdR.

Publications

Transcriptional activation of the bkd operon of Pseudomonas putida by BkdR.

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