Relevance of nucleotides of the PcaU binding site from Acinetobacter baylyi

Jerg, Bettina; Gerischer, Ulrike

doi:10.1099/mic.0.2007/013508-0

Cited by 7 publications

(8 citation statements)

References 30 publications

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“…If this set of nucleotides were those directly bound by TtgV, this may explain why the affinity of TtgV for the ttgD operator is higher and correlates with the lower expression of the ttgDEF operon in vivo. Long palindromic DNA targets for IclR family members have been described recently for Streptomyces CcaR, a regulator involved in the control of ␤-lactam antibiotics in this microorganism (25), and Acinetobacter PcaU, a regulator involved in the metabolism of aromatic carboxylic acids (7).…”

Section: Discussionmentioning

confidence: 99%

TtgV Represses Two Different Promoters by Recognizing Different Sequences

et al. 2009

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Expression of the multidrug efflux pump ttgDEF and ttgGHI operons is modulated in vivo mainly by the TtgV repressor. TtgV is a multidrug recognition repressor that exhibits a DNA binding domain with a long interaction helix comprising residues 47 to 64. The pattern of expression of the two pumps is different in Pseudomonas putida: in the absence of effectors, the promoter for the ttgD gene is silent, whereas the ttgG gene is expressed at a high basal level. This correlates with the fact that TtgV exhibits a higher affinity for the ttgD operator (K D ‫؍‬ 10 ؎ 1 nM) than for the ttgG (K D ‫؍‬ 19 ؎ 1 nM) operator. Sequence analysis revealed that both operators are 40% identical, and mutational analysis of the ttgD and ttgG operators combined with electrophoretic mobility shift assays and in vivo expression analysis suggests that TtgV recognizes an inverted repeat with a high degree of palindromicity around the central axis. We generated a collection of alanine substitution mutants with substitutions between residues 47 and 64 of TtgV. The results of extensive combinations of promoter variants with these TtgV alanine substitution mutants revealed that TtgV modulates expression from ttgD and ttgG promoters through the recognition of both common and different sequences in the two promoters. In this regard, we found that TtgV mutants at residues 48, 50, 53, 54, 60, and 61 failed to bind ttgG but recognized the ttgD operator. TtgV residues R47, R52, L57, and T49 are critical for binding to both operators. Based on three-dimensional models, we propose that these residues contact nucleotides within the major groove of DNA.

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Section: Discussionmentioning

confidence: 99%

TtgV Represses Two Different Promoters by Recognizing Different Sequences

et al. 2009

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“…In the presence of the inducer protocatechuate, PcaU brings about high induction and is thus both a repressor and an activator (47, 53). PcaU is an IclR family member and binds to a site between the pca-qui genes and its own gene containing three repetitions of a 10-bp DNA sequence, which are all necessary for induction (29,36). Additional regulatory levels of higher priority can prevent induction despite the continued presence of the inducer (Fig.…”

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confidence: 99%

Role of Acinetobacter baylyi Crc in Catabolite Repression of Enzymes for Aromatic Compound Catabolism

et al. 2009

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Here, we describe for the first time the Crc (catabolite repression control) protein from the soil bacterium Acinetobacter baylyi. Expression of A. baylyi crc varied according to the growth conditions. A strain with a disrupted crc gene showed the same growth as the wild type on a number of carbon sources. Carbon catabolite repression by acetate and succinate of protocatechuate 3,4-dioxygenase, the key enzyme of protocatechuate breakdown, was strongly reduced in the crc strain, whereas in the wild-type strain it underwent strong catabolite repression. This strong effect was not based on transcriptional regulation because the transcription pattern of the pca-qui operon (encoding protocatechuate 3,4-dioxygenase) did not reflect the derepression in the absence of Crc. pca-qui transcript abundance was slightly increased in the crc strain. Lack of Crc dramatically increased the mRNA stability of the pca-qui transcript (up to 14-fold), whereas two other transcripts (pobA and catA) remained unaffected. p-Hydroxybenzoate hydroxylase activity, encoded by pobA, was not significantly different in the absence of Crc, as protocatechuate 3,4-dioxygenase was. It is proposed that A. baylyi Crc is involved in the determination of the transcript stability of the pca-qui operon and thereby effects catabolite repression.The introduction of aromatic compounds into the central energy conservation pathways is accomplished in Acinetobacter baylyi via the ␤-ketoadipate pathway (25). In Acinetobacter, this pathway contains two parallel branches separated in terms of enzymes, as well as regulation of expression, converting the two starting compounds protocatechuate and catechol into succinyl coenzyme A and acetyl coenzyme A (21). Protocatechuate breakdown requires six catalytic steps; the respective genes (pca genes), together with genes for one of several funneling pathways (qui genes), form a large operon (the pca-qui operon, about 14 kbp) (10). Regulation of the expression of this operon is directed from the intergenic region located upstream of this large gene cluster. Several levels of transcriptional regulation of pca-qui gene expression have been described, most of which have a negative effect and therefore serve to prevent gene expression. Only one mechanism causes induction at the otherwise weak promoter upstream of pcaI (pcaIp), namely, the activity of the regulator PcaU (22). In its absence, the pca-qui genes are expressed at a fairly high basal level. PcaU decreases this basal expression level. In the presence of the inducer protocatechuate, PcaU brings about high induction and is thus both a repressor and an activator (47, 53). PcaU is an IclR family member and binds to a site between the pca-qui genes and its own gene containing three repetitions of a 10-bp DNA sequence, which are all necessary for induction (29,36). Additional regulatory levels of higher priority can prevent induction despite the continued presence of the inducer (Fig. 1). One is a mechanism that seems to organize gene expression priorities between the two ...

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“…This confirmed that HisTphR II directly binds to the region from positions Ϫ66 to Ϫ36, which contains an inverted repeat sequence, 5Ј-TTTTTGCGC ATAGCGCAAAAA-3Ј, centered at position Ϫ51 from the tphC II transcription start site. In the case of the binding sites for several ITTRs, including PcaU, PcaR, and PobR, an external direct sequence repetition of the half site of the inverted repeat sequences was observed (7,11,16,28); and this direct sequence repetition was required for the binding of PcaU (28). No such direct repeat sequence was found in the tphR II -tphC II intergenic region.…”

Section: Resultsmentioning

confidence: 97%