scholarly journals Clp-dependent proteolysis of the LexA N-terminal domain in Staphylococcus aureus

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Marianne T. Cohn ◽  
Peter Kjelgaard ◽  
Dorte Frees ◽  
José R. Penadés ◽  
Hanne Ingmer
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Bacterial Species ◽  
Transcriptional Regulator ◽  
Sos Response ◽  
Binding Activity ◽  
Dimerization Domain ◽  
Dna Binding Activity ◽  
Lexa Repressor ◽  
Induced Mutagenesis

The SOS response is governed by the transcriptional regulator LexA and is elicited in many bacterial species in response to DNA damaging conditions. Induction of the SOS response is mediated by autocleavage of the LexA repressor resulting in a C-terminal dimerization domain (CTD) and an N-terminal DNA-binding domain (NTD) known to retain some DNA-binding activity. The proteases responsible for degrading the LexA domains have been identified in Escherichia coli as ClpXP and Lon. Here, we show that in the human and animal pathogen Staphylococcus aureus, the ClpXP and ClpCP proteases contribute to degradation of the NTD and to a lesser degree the CTD. In the absence of the proteolytic subunit, ClpP, or one or both of the Clp ATPases, ClpX and ClpC, the LexA domains were stabilized after autocleavage. Production of a stabilized variant of the NTD interfered with mitomycin-mediated induction of sosA expression while leaving lexA unaffected, and also significantly reduced SOS-induced mutagenesis. Our results show that sequential proteolysis of LexA is conserved in S. aureus and that the NTD may differentially regulate a subset of genes in the SOS regulon.

scholarly journals The glucosaminidase domain of Atl – the major Staphylococcus aureus autolysin – has DNA ‐binding activity

2014 ◽  
Vol 3 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Inês R. Grilo ◽  
Ana Madalena Ludovice ◽  
Alexander Tomasz ◽  
Hermínia Lencastre ◽  
Rita G. Sobral
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Binding Activity ◽  
Dna Binding Activity

scholarly journals Identification of a Novel ENU-Induced Mutation in Mouse Tbx1 Linked to Human DiGeorge Syndrome

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Jiaofeng Chen ◽  
Xue Zhang ◽  
Jie Li ◽  
Chenmeng Song ◽  
Yichang Jia ◽  
...  
Keyword(s):  
Dna Binding ◽  
Digeorge Syndrome ◽  
Hearing Threshold ◽  
Chronic Otitis Media ◽  
Screening Strategy ◽  
Binding Activity ◽  
Nonsynonymous Mutation ◽  
T Box ◽  
Dna Binding Activity ◽  
Induced Mutagenesis

The patients with DiGeorge syndrome (DGS), caused by deletion containing dozens of genes in chromosome 22, often carry cardiovascular problem and hearing loss associated with chronic otitis media. Inside the deletion region, a transcription factor TBX1 was highly suspected. Furthermore, similar DGS phenotypes were found in the Tbx1 heterozygous knockout mice. Using ENU-induced mutagenesis and G1 dominant screening strategy, here we identified a nonsynonymous mutation p.W118R in T-box of TBX1, the DNA binding domain for transcription activity. The mutant mice showed deficiency of inner ear functions, including head tossing and circling, plus increased hearing threshold determined by audiometry. Therefore, our result further confirms the pathogenic basis of Tbx1 in DGS, points out the crucial role of DNA binding activity of TBX1 for the ear function, and provides additional animal model for studying the DGS disease mechanisms.

scholarly journals Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus

2001 ◽  
Vol 183 (10) ◽  
pp. 3016-3024 ◽  
Author(s):  
Frank S. Kaczmarek ◽  
Richard P. Zaniewski ◽  
Thomas D. Gootz ◽  
Dennis E. Danley ◽  
Mahmoud N. Mansour ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Dna Binding ◽  
Functional Characterization ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Dna Ligase ◽  
E Coli ◽  
Dna Binding Activity ◽  
C Terminus

ABSTRACT A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD+-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD+-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD+-dependent DNA ligase fromB. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

scholarly journals Study on DNA-binding Activity of Heme-containing Transcriptional Regulator CooA by Using Fluorescence Anisotropy

2000 ◽  
Vol 40 (supplement) ◽  
pp. S93
Author(s):  
K. Yamamoto ◽  
H. Ishikawa ◽  
K. Ishimori ◽  
I. Morishima ◽  
H. Nakajima ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fluorescence Anisotropy ◽  
Transcriptional Regulator ◽  
Binding Activity ◽  
Dna Binding Activity

scholarly journals The E2A-HLF Oncoprotein ActivatesGroucho-Related Genes and SuppressesRunx1

2001 ◽  
Vol 21 (17) ◽  
pp. 5935-5945 ◽  
Author(s):  
Jinjun Dang ◽  
Takeshi Inukai ◽  
Hidemitsu Kurosawa ◽  
Kumiko Goi ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Fusion Gene ◽  
Chromosomal Translocation ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Representational Difference Analysis ◽  
Transactivation Domain ◽  
Dimerization Domain ◽  
Dna Binding Activity

ABSTRACT The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells, encodes a chimeric transcription factor consisting of the transactivation domain of E2A linked to the bZIP DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF). This oncoprotein blocks apoptosis induced by growth factor deprivation or irradiation, but the mechanism for this effect remains unclear. We therefore performed representational difference analysis (RDA) to identify downstream genetic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were differentially upregulated in E2A-HLF-positive cells after zinc induction. The corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptional corepressor that lacks DNA-binding activity on its own but can act in concert with other proteins to regulate embryologic development of the fly. Expression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected increased amounts of two additional Groucho-related proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell survival and activated Groucho-related genes. Among the transcription factors known to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis.

scholarly journals A novel redox-sensing transcriptional regulator CyeR controls expression of an Old Yellow Enzyme family protein in Corynebacterium glutamicum

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1335-1341 ◽  
Author(s):  
Shigeki Ehira ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Oxidative Stress ◽  
Dna Binding ◽  
Corynebacterium Glutamicum ◽  
Redox Regulation ◽  
Transcriptional Regulator ◽  
Binding Activity ◽  
Old Yellow Enzyme ◽  
Dna Binding Activity ◽  
Family Protein ◽  
Redox Sensing

Corynebacterium glutamicum cgR_2930 (cyeR) encodes a transcriptional regulator of the ArsR family. Its gene product, CyeR, was shown here to repress the expression of cyeR and the cgR_2931 (cye1)–cgR_2932 operon, which is located upstream of cyeR in the opposite orientation. The cye1 gene encodes an Old Yellow Enzyme family protein, members of which have been implicated in the oxidative stress response. CyeR binds to the intergenic region between cyeR and cye1. Expression of cyeR and cye1 is induced by oxidative stress, and the DNA-binding activity of CyeR is impaired by oxidants such as diamide and H2O2. CyeR contains two cysteine residues, Cys-36 and Cys-43. Whereas mutation of the former (C36A) has no effect on the redox regulation of CyeR activity, mutating the latter (C43A, C43S) abolishes the DNA-binding activity of CyeR. Cys-43 of CyeR and its C36A derivative are modified upon treatment with diamide, suggesting an important role for Cys-43 in the redox regulation of CyeR activity. It is concluded that CyeR is a redox-sensing transcriptional regulator that controls cye1 expression.

scholarly journals Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

2019 ◽  
Author(s):  
Leah M. Williams ◽  
Melissa M. Inge ◽  
Katelyn M. Mansfield ◽  
Anna Rasmussen ◽  
Jamie Afghani ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Functional Characterization ◽  
Binding Activity ◽  
Sponge Tissue ◽  
Dimerization Domain ◽  
Dna Binding Activity ◽  
Iκb Kinase

ABSTRACTBiological and biochemical functions of immunity transcription factor NF-κB in basal metazoans are largely unknown. Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq), in the phylum Porifera. Structurally and phylogenetically, the Aq-NF-κB protein is most similar to NF-κB p100 and p105 among vertebrate proteins, with an N-terminal DNA-binding/dimerization domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile more similar to human NF-κB proteins than Rel proteins. Aq-NF-κB also resembles the mammalian NF-κB protein p100 in that C-terminal truncation results in translocation of Aq-NF-κB to the nucleus and increases its transcriptional activation activity. Overexpression of a human or sea anemone IκB kinase (IKK) can induce C-terminal processing of Aq-NF-κB in vivo, and this processing requires C-terminal serine residues in Aq-NF-κB. Unlike human NF-κB p100, however, the C-terminal sequences of Aq-NF-κB do not effectively inhibit its DNA-binding activity when expressed in human cells. Tissue of another demosponge, a black encrusting sponge, contains NF-κB site DNA-binding activity and an NF-κB protein that appears mostly processed and in the nucleus of cells. NF-κB DNA-binding activity and processing is increased by treatment of sponge tissue with LPS. By transcriptomic analysis of A. queenslandica we identified likely homologs to many upstream NF-κB pathway components. These results present a functional characterization of the most ancient metazoan NF-κB protein to date, and show that many characteristics of mammalian NF-κB are conserved in sponge NF-κB, but the mechanism by which NF-κB functions and is regulated in the sponge may be somewhat different.

scholarly journals Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
César L. Pastrana ◽  
Ngat T. Tran ◽  
Clare. E. Stevenson ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

ABSTRACTThe tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus.

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