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Publication History View all

  • Cell cycle (Georgetown, Tex.) 11/2013; 13(1).
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    ABSTRACT: The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.
    Nature Communications 11/2013; 4:2796.
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    ABSTRACT: Hydrolysis of the phosphodiester bonds of the transcript by bacterial RNA polymerase is assisted by 3'NMP of the RNA. Here we provide evidence that this mechanism is also involved in RNA cleavage by eukaryotic RNA polymerase II, suggesting that transcript assisted hydrolysis has emerged before divergence of bacteria and archaea/eukaryotes.
    Transcription. 11/2013; 4(5).
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    ABSTRACT: Insertion of an apramycin resistance cassette in the clpP1clpP2 operon (encoding the ClpP1 and ClpP2 peptidase subunits) affects morphological and physiological differentiation of Streptomyces lividans. Another key factor controlling Streptomyces differentiation is the pleiotropic transcriptional regulator AdpA. We have identified a spontaneous missense mutation (-1 frameshift) in the adpA (bldH) open reading frame in a clpP1clpP2 mutant that led to the synthesis of a non-functional AdpA protein. Electrophoretic mobility shift assays showed that AdpA bound directly to clpP1clpP2 promoter region. Quantitative real-time PCR analysis showed that AdpA regulated the clpP1clpP2 operon expression at specific growth times. In vitro, AdpA and ClgR, a transcriptional activator of clpP1clpP2 operon and other genes, were able to bind simultaneously to clpP1 promoter, which suggests that AdpA binding to clpP1 promoter did not affect that of ClgR. This study allowed to uncover an interplay between the ClpP peptidases and AdpA in S. lividans.
    Archives of Microbiology 11/2013;
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    ABSTRACT: Bacterial cell division is a highly coordinated process that begins with the polymerization of the tubulin-like protein FtsZ at midcell. FtsZ polymerization is regulated by a set of conserved cell division proteins, including ZapA. However, a zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study, we used a synthetic-lethal screen to find genes that become essential when ZapA is mutated. Three transposon insertions were found in yvcL. Deletion of yvcL in a wild type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ-ring assembly, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25% identical and 50% similar to the Streptomyces coelicolor transcription factor WhiA, which induces ftsZ and is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP-on-chip assay gave no indication that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a yvcL zapA double mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implicated in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as yet unknown mechanism.
    Journal of bacteriology 10/2013;
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    ABSTRACT: HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.
    Molecular cell 10/2013;
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    ABSTRACT: The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms (SNPs) were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen (PA), the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell wall synthesis, resulting in the induction of the cell wall stress regulon that encodes another quality control protease. This article is protected by copyright. All rights reserved.
    Proteomics 09/2013;
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    ABSTRACT: Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.
    Cell 08/2013; 154(5):1140-50.
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    ABSTRACT: Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (∼1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce backtracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.
    Nucleic Acids Research 08/2013;
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    ABSTRACT: Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique crosslinking assay that specifically detects ATP-dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Eschericia coli DnaA where ATP binding appears to be the targeted activity.
    Molecular Microbiology 08/2013;
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