Brahma Is Required for Proper Expression of the Floral Repressor FLC in Arabidopsis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development. [Methodology/Principal Findings]: Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable. [Conclusions/Significance]: BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.This work was supported by Ministerio de Educacin y Ciencia (BFU2008-00238, CSD2006-00049), and by Junta de Andaluca (P06-CVI-01400) to J.C.R. and by the National Institutes of Health (grant no. 1R01GM079525), and the National Science Foundation (grant no. 0446440) to R.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Bacterial Toxin-Antitoxin Systems’ Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes’ expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage

The Axe-Txe Complex of Enterococcus faecium Presents a Multilayered Mode of Toxin-Antitoxin Gene Expression Regulation

Multidrug-resistant variants of human pathogens from the genus Enterococcus represent a significant health threat as leading agents of nosocomial infections. The easy acquisition of plasmid-borne genes is intimately involved in the spread of antibiotic resistance in enterococci. Toxin-antitoxin (TA) systems play a major role in both maintenance of mobile genetic elements that specify antibiotic resistance, and in bacterial persistence and virulence. Expression of toxin and antitoxin genes must be in balance as inappropriate levels of toxin can be dangerous to the host. The controlled production of toxin and antitoxin is usually achieved by transcriptional autoregulation of TA operons. One of the most prevalent TA modules in enterococcal species is axe-txe which is detected in a majority of clinical isolates. Here, we demonstrate that the axe-txe cassette presents a complex pattern of gene expression regulation. Axe-Txe cooperatively autorepress expression from a major promoter upstream of the cassette. However, an internal promoter that drives the production of a newly discovered transcript from within axe gene combined with a possible modulation in mRNA stability play important roles in the modulation of Axe:Txe ratio to ensure controlled release of the toxin

The role of a putative terminator region downstream of the <i>txe</i> gene.

<p>(<b>A</b>) <i>E. coli</i> SC301467 harbouring derivatives of pTE103 bearing the <i>axe-txe</i> cassette with (pat_axe-txe_ter) or without (pat_axe-txe) the putative downstream transcription terminator were grown at 37⁰C. Absorbance readings at 600 nm were taken at 60 minutes intervals. (<b>B</b>) The terminator in the region downstream of the <i>txe</i> gene was predicted and drawn by the MFOLD program.</p

An active <i>p</i>_<i>axe</i> promoter is required for <i>axe-txe</i> mediated stable plasmid maintenance.

<p>Stability assays were conducted with derivatives of the stability probe vector, pFH450: pREGΔaxe-txe does not contain any accessory stability determinants (circles), pREG531 contains the <i>axe-txe</i> cassette (squares), and pREGpaxemut contains the <i>axe-txe</i> cassette with a mutated <i>p</i>_<i>axe</i> promoter (triangles). Assays were performed as outlined in Materials and Methods. Results are averages of at least five experiments for which the standard deviation did not exceed 15%.</p

Axe and Axe-Txe binding to the <i>p</i>_<i>at</i> promoter-operator region.

<p>A 295-bp 5’ biotinylated fragment that included the <i>axe</i> translation start codon and upstream promoter-operator region was subjected to EMSA. The fragment was incubated with different concentrations of <i>E. coli</i> BL21(DE3) crude extracts (left to right in each panel): 0, 1.25, 2.5, 5, 10, 12.5 and 25 µg/ml. Reactions were incubated for 20 min at 22⁰C, analyzed by native 5% PAGE, and processed further as outlined in Materials and Methods. (<b>A</b>) no Axe or Txe produced; (<b>B</b>) Axe overproduction; (<b>C</b>) Axe-Txe overproduction. Filled and open arrows denote positions of unbound DNA and protein-DNA complexes, respectively.</p

<i>P</i>_<i>axe</i> promoter sequence and activity.

<p>(<b>A</b>) Nucleotide sequence of the <i>p</i>_<i>axe</i> region. The transcription start site mapped by primer extension is marked by a vertical arrow. -10 and -35 promoter motifs are underlined and the <i>txe</i> start codon is in bold. (<b>B</b>) Primer extension analysis of <i>p</i>_<i>axe</i>. Total RNA from <i>E. coli</i> SC301467 cells harbouring a plasmid possessing the <i>axe</i> gene was subjected to primer extension analysis (E) using a radioactively labelled primer that anneals within flanking vector sequences. Reactions were performed and analysed as outlined in Materials and Methods, and electrophoresed on a denaturing 6% polyacrylamide gel in parallel with nucleotide sequencing reactions (A, C, G, T) carried out with the same primer. The major product from the primer extension is marked as +1. (<b>C</b>) A transcriptional fusion of the <i>axe</i> gene to the <i>luxCDABE</i> operon in pBBRlux-amp plasmid (paxe_lux) was transformed into <i>E. coli</i> SC301467 and luminescence in RLU (relative luminescence units) determined. paxemut_lux denotes a construct in which <i>p</i>_axe possesses two substitution mutations in the -10 box (see text). The results are the averages of at least three independent experiments.</p

A fragment downstream of <i>txe</i> acts as a putative transcriptional terminator/attenuator <i>in vitro</i>.

<p>Multi-round <i>in vitro</i> transcription experiments were performed using <i>E. coli</i> σ⁷⁰ RNA polymerase holoenzyme and pTE103 template DNAs containing the whole <i>axe-txe</i> operon fragment (1), the same fragment but with the <i>p</i>_<i>axe</i> promoter mutated (2), or the whole <i>axe-txe</i> operon fragment plus the downstream putative terminator region (3). Reactions were performed and analysed as outlined in Materials and Methods. Transcript sizes were estimated according to an RNA ladder (RiboRuler Low Range RNA Ladder – Thermo Scientific) which was electrophoresed with the reactions and then excised and stained with ethidium bromide. Positions corresponding to the RNA ladder bands are marked at the right site of the autoradiogram (L). Sizes and schematic representation of the transcripts with the terminator hairpins (“peaks”) are drawn on the left site of the figure.</p

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication