Connecting primary and secondary metabolism: AreB, an IclR‐like protein, binds the AREccaR sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production

Santamarta, Irene; López-García, María Teresa; Pérez‐Redondo, Rosario; Koekman, Bert; Martı́n, Juan F.; Liras, Paloma

doi:10.1111/j.1365-2958.2007.05937.x

Cited by 37 publications

(34 citation statements)

References 40 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 chromosome, ndgR is positioned adjacent to leuCD encoding isopropylmalate dehydrogenase subunits. Two independent studies with S. clavuligerus and S. coelicolor demonstrated that NdgR positively regulated leuCD expression (26,31). However, knockout of ndgR and its ortholog had far-reaching impacts on the production of antibiotics such as ACT-undecylprodigiosin (Red) and clavulanic acid-cephamycin.…”

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confidence: 99%

NdgR, a Common Transcriptional Activator for Methionine and Leucine Biosynthesis in Streptomyces coelicolor

et al. 2012

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We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts.

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“…Although it is very important to understand how gene regulators control primary and secondary metabolism, there have been only a few studies on regulators directly linking primary and secondary metabolism in S. coelicolor (30). In addition, there are not many reports on regulators that simultaneously control primary metabolism as well as antibiotic production by directly binding to corresponding promoter regions such as actII-ORF4, cdaR, and redZ (22).…”

Section: Discussionmentioning

confidence: 99%

Rapid Functional Screening of Streptomyces coelicolor Regulators by Use of a pH Indicator and Application to the MarR-Like Regulator AbsC

Yang

Song

Lee

et al. 2010

Appl Environ Microbiol

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To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production between a deletion mutant and a wild-type strain (WT) were compared. These differences are easily hidden by complex media. To determine the specific nutrient conditions that reveal such differences, we used a multiwell method containing different nutrients along with bromothymol blue. We found several nutrients that provide key information on characterization conditions. By comparing the growth of wild-type and mutant strains on screened nutrients, we were able to measure growth, organic acid production, and antibiotic production for the elucidation of regulator function. As a result of this method, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and ␣-ketoglutarate and increased extracellular actinorhodin (ACT) production in minimal medium containing glucose and alanine (MMGA). This method could therefore be a high-throughput method for the characterization of unknown regulators.The rapid increase in genome sequencing data, combined with proteomics techniques and a PCR targeting method using undecylprodigiosin ( RED)-mediated recombination (12), has made available abundant physiological and genetic information on Streptomyces species, the major producers of antibiotics (2, 16). These technical advances are time-saving with the help of bioinformatics and therefore can contribute to elucidating the individual and exact functions of genes (8). Despite these developments, the rapid functional identification of any unknown gene is still difficult and time-consuming when the gene is located between uncharacterized, neighboring genes or when it does not show any homology to other species. Especially for Streptomyces, which has many more regulators than any other bacterium (42), the existence of unknown or putative regulators prevents the full understanding of antibiotic production and regulator interaction under various conditions. Even worse, detection of differences in gene expression levels is difficult, since Streptomyces bacteria express biosynthetic and catabolic enzymes at relatively low constitutive levels (15). Thus, elucidating the relationship between regulatory function and nutrient composition takes more time than finding novel regulators using various approaches.One of the easiest ways to characterize the function of a regulator is to delete the gene and monitor differences that occur in phenotype or antibiotic production compared to those of the wild type (WT) under various nutrient conditions. This is effective because the primary and secondary metabolisms of Streptomyces are tremendously affected by nutrients, as are various other cellular events (27, 28) which involve many regulators either directly and/or indirectly ( Fig. 1) (42). However, there are also many regulator...

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Connecting primary and secondary metabolism: AreB, an IclR‐like protein, binds the ARE_ccaR sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production

Cited by 37 publications

References 40 publications

TtgV Represses Two Different Promoters by Recognizing Different Sequences

TtgV Represses Two Different Promoters by Recognizing Different Sequences

NdgR, a Common Transcriptional Activator for Methionine and Leucine Biosynthesis in Streptomyces coelicolor

Rapid Functional Screening of Streptomyces coelicolor Regulators by Use of a pH Indicator and Application to the MarR-Like Regulator AbsC

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