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Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168.

ABSTRACT: Bacillus subtilis 168 is resistant to phenolic acids by expression of an inducible enzyme, the phenolic acid decarboxylase (PadC), that decarboxylates these acids into less toxic vinyl derivatives. In the phenolic acid stress response (PASR), the repressor of padC, PadR, is inactivated by these acids. Inactivation of PadR is followed by a strong expression of padC. To elucidate the functional interaction between PadR and the padC promoter, we performed (i) footprinting assays to identify the region protected by PadR, (ii) electrophoretic mobility shift assays (EMSAs) with a modified padC promoter protected region to determine the interacting sequences, and (iii) random mutagenesis of padR to identify amino acid residues essential for the function of PadR. We identified an important consensus dyad sequence called IR1-2 (ATGT-8N-ACAT) overlapping a second dyad element (GTGT-8N-ACAT) that we named dIR1-2bis. The entire dIR1-2bis/IR1-2 sequence permits binding of two PadR dimers in EMSAs, which may be observed for bacteria grown under noninduced conditions where the padC promoter is completely repressed. Three groups of modified PadRs giving a PASR phenotype were characterized in vivo. The DNA sequences of certain mutant padR alleles indicate that important residues are all located in the region containing the coiled-coil leucine zipper domain that is involved in dimerization. These substitutions reduce the affinity of PadR binding to the padC promoter. Of particular interest are residue L128, located at the center of the putative coiled-coil leucine zipper domain, and residue E97, which is conserved among all PadRs.

SUBMITTER: Nguyen TK

PROVIDER: S-EPMC3147689 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168.

Nguyen Thi Kim Chi TK Tran Ngoc Phuong NP Cavin Jean-François JF

Journal of bacteriology 20110617 16

Bacillus subtilis 168 is resistant to phenolic acids by expression of an inducible enzyme, the phenolic acid decarboxylase (PadC), that decarboxylates these acids into less toxic vinyl derivatives. In the phenolic acid stress response (PASR), the repressor of padC, PadR, is inactivated by these acids. Inactivation of PadR is followed by a strong expression of padC. To elucidate the functional interaction between PadR and the padC promoter, we performed (i) footprinting assays to identify the reg ...[more]

PMID: 21685295

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Project description:Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.

Project description:BackgroundButanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities.ResultsUsing comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline-glutamate-arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested.ConclusionThis work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.

Dataset Information

Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168.

Publications

Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168.

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