Transcription regulation in thermophilic Bacteria: high resolution contact probing of Bacillus stearothermophilus and Thermotoga neapolitana arginine repressor-operator interactions

Song, Hui; Wang, Haifeng H.; Gigot, Daniel; Dimova, Diliana; Sakanyan, Vehary; Glansdorff, Nicolas; Charlier, Daniël

doi:10.1006/jmbi.2001.5236

Cited by 27 publications

(39 citation statements)

References 56 publications

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“…The ARG boxes of L. lactis differ from those of most other systems by the presence of a large interoperator spacer region. Such spacer regions are generally 2-3 bp in E. coli, B. stearothermophilus, B. subtilis, and Thermotoga neapolitana (8,21,25), compared with 32 bp for the PargC operators and possibly 75 and 10 bp for the PgltS and PargG operators, respectively (with ARG box lengths of 18 bp). No clear difference in affinity of the regulators for argC O1 and argC O2 was apparent, and the presence of hypersensitive residues in the DNA footprint of the PargC region between the two operators suggests that DNA bending takes place.…”

Section: Discussionmentioning

confidence: 99%

“…Despite differences in the organization of genes involved in arginine metabolism, experimental evidence indicates that the mechanism of arginine-dependent regulation of these genes is highly conserved among a range of different organisms, including Gram-negative, Gram-positive and extremophilic bacteria (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Regulation is exerted by binding of single transcriptional regulators of the ArgR family to so-called ARG operator sites preceding the relevant target genes, generally leading to repression of arginine biosynthetic genes and activation of catabolic genes, in the presence of arginine.…”

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confidence: 99%

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Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

Larsen

Kok

Kuipers

2005

Journal of Biological Chemistry

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The expression of arginine metabolism in Lactococcus lactis is controlled by the two homologous transcriptional regulators ArgR and AhrC. Genome sequence analyses have shown that the occurrence of multiple homologues of the ArgR family of transcriptional regulators is a common feature of many low-G ؉ C Gram-positive bacteria. Detailed studies of ArgR type regulators have previously only been carried out in bacteria containing single regulators. Here, we present a first characterization of the two L. lactis arginine regulators by means of gel retardation and DNase I footprinting. ArgR of L. lactis was shown to bind to the promoter regions of both the arginine biosynthetic argCJDBF operon and the arginine catabolic arcABD1C1C2TD2yvaD operon, but in an arginine-independent manner. Surprisingly, AhrC alone was unable to bind to DNA. Arginine-dependent DNA binding was obtained by mixing the two regulators in gel retardation assays. With both regulators present, the addition of arginine led to increased binding of ArgR-AhrC to the biosynthetic argC promoter but also to diminished binding to the catabolic arcA promoter. Footprinting showed ArgR-AhrC protection of regions containing ARG box operator sequences preceding argC. In the absence of AhrC, ArgR protected sites in the arcA promoter region with similarity to ARG box half-sites, here called ARC boxes. We propose a model for repression of arginine biosynthesis and activation of catabolism by anti-repression, involving arginine-dependent interaction between the two L. lactis regulator proteins, ArgR and AhrC.Despite differences in the organization of genes involved in arginine metabolism, experimental evidence indicates that the mechanism of arginine-dependent regulation of these genes is highly conserved among a range of different organisms, including Gram-negative, Gram-positive and extremophilic bacteria (1-12). Regulation is exerted by binding of single transcriptional regulators of the ArgR family to so-called ARG operator sites preceding the relevant target genes, generally leading to repression of arginine biosynthetic genes and activation of catabolic genes, in the presence of arginine.Crystal structures of the ArgR type transcriptional regulators of Escherichia coli (ArgREc (13, 14)), Bacillus stearothermophilus (ArgRBst (15)), and Bacillus subtilis (AhrCBsu (16)) have revealed these to be structurally similar proteins, making up a complex of six identical subunits. The subunits are arranged in hexameric structures, which are organized as dimers of trimers. In E. coli and B. subtilis, the hexameric structure is maintained both in the absence and presence of arginine (4, 17), whereas the regulator of B. stearothermophilus mainly exists as a trimer that assembles into hexamers dependent of the concentrations of arginine, protein, and DNA (5, 15). Six arginine molecules are bound at the trimer-trimer interface, strengthening the interaction between the trimers and at the same time introducing a conformational change in the regulator, thus increasing its affini...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

Larsen

Kok

Kuipers

2005

Journal of Biological Chemistry

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“…The true mechanism for heat denaturation is probably hidden in HrcA molecules complexed with the CIRCE element. Studies of other repressors from thermophilic microorganisms have been reported (4,11,24). The thermostabilities of the DNA-binding protein HU from mesophilic, thermophilic, and extremely thermophilic bacteria were compared, and Christodoulou and Vorgias concluded that local interactions at the HTH region are responsible for the thermostabilities of HU proteins (4).…”

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Identification of a Helix-Turn-Helix Motif of Bacillus thermoglucosidasius HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor

et al. 2003

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In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.In the heat shock response of Bacillus subtilis, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element (9, 17). Studies of HrcA and hrcA, its gene in various microorganisms, have clarified that HrcA molecules have similar features, e.g., the primary sequences of HrcA are strikingly conserved among organisms; based on phylogenetic analysis, this characteristic has evolutionary implications (1,22,33). On the other hand, common and unfavorable characteristics are that HrcA is hardly soluble and easily forms aggregates. This disadvantage has prevented the clarification of the real nature of HrcA. To circumvent this disadvantage, it was demonstrated in a previous report that HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006 was most efficiently renatured by the addition of DNA, including the CIRCE element, and that the HrcA-CIRCE complex was stable in the soluble form in the absence of GroEL (29).Two critical issues related to the structure and function of HrcA are outstanding. One is the identification of the region at which HrcA binds to the CIRCE element. Although three conserved regions have been previously pointed out (22,33), no extended analysis of the DNA-binding region of HrcA has been conducted. The other issue is whether HrcA can play the role of a thermosensor. When bacillary cells are exposed to heat stress, the expression of genes controlled by the HrcA-CIRCE complex is turned on. This implies that the HrcA-CIRCE complex must be decomposed by heat stress to release the regulation system. However, no direct evidence that the HrcA-CIRCE complex is dec...

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“…The ArgR repressor binds a 42-bp operator comprising two Arg box-like sequences separated by a 2-bp spacer and overlapping the PargC promoter located upstream of the argCJBD operon (10,35). The three-dimensional structure of a full-length aporepressor and argininebound C-terminal domain of B. stearothermophilus ArgR has recently been resolved (28).…”

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confidence: 99%

“…The C-terminal His tag is shown in lowercase letters. Dashed brackets indicate operator sequences protected against DNase I cleavage upon wild-type repressor binding (35,46). Spacer nucleotides between two Arg boxes are shown in lowercase letters.…”

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Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

et al. 2002

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Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the ␣4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the ␣4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.Recent genome comparative analysis has revealed a global vision of arg genes transcription regulation in microorganisms (3,23,26,30). However, experimental studies on distant organisms are required to elucidate the molecular mechanisms and to understand the evolutionary principles established for arginine regulatory systems in various microbes.A substantial amount of functional and structural information regarding transcription regulation has accumulated for the Escherichia coli arginine repressor, ArgR (14,22,40). In this organism, the repressor, in cooperation with L-arginine, governs expression of the arginine biosynthesis genes (arg) and carAB genes, coding for carbamoylphosphate synthetase (EC 6.3.5.5), providing the carbamoylphosphate required for the synthesis of both arginine and pyrimidine residues. The ArgR protein consists of N-terminal DNA-binding and C-terminal oligomerization domains connected by a short protease-sensitive linker (15), and a three-dimensional structure has been separately resolved for each domain (41,45). A winged helixturn-helix (wHTH) motif of the DNA-binding domain (41) recognizes a 40-bp sequence which comprises two adjacent imperfect 18-bp palindromes, known as Arg boxes, that are separated by a 3-bp spacer in the majority of cognate operators or by a 2-bp spacer in the argR operator (6,44,46). ArgR monomers associ...

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Transcription regulation in thermophilic Bacteria: high resolution contact probing of Bacillus stearothermophilus and Thermotoga neapolitana arginine repressor-operator interactions

Cited by 27 publications

References 56 publications

Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

Identification of a Helix-Turn-Helix Motif of Bacillus thermoglucosidasius HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor

Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors fromEscherichia coliandBacillus stearothermophilus

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