Search

A position sensitive detector (PSD) adapted to the technical and mechanical specifications of our angle and energy resolved electron-ion(s) coincidence experiments is described in this article. The device, whose principle is very similar to the one detailed by J. H. D. Eland [Meas. Sci. Technol. 5, 1501 (1994)], is composed by a set of microchannel plates and a delay line anode. The originality comes from the addition in front of the encoding surface of a ceramic disk covered by a resistive surface. The capacitive coupling between the anode and the resistive plane has the double advantage of eliminating the spatial modulations due to the lattice of the anode and also of sensitizing a greater number of electrodes, increasing thus considerably the accuracy of the position measurements. The tests carried out with a time to digital conversion module of 250 ps resolution showed that a spatial resolution better than 50 μm and a dead time of 160 ns can be achieved. Typical images obtained with the help of the EPICEA and DELICIOUS coincidence setups are also shown. [ABSTRACT FROM AUTHOR]

Expression of the mouse cardiac actin gene depends on a distal enhancer (−7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3′ AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation. [ABSTRACT FROM AUTHOR]

This paper describes changes in the dimensions of fibres in the lungs of rats and in fibres removed from the lungs by broncho-alveolar lavage, following a single inhalation exposure to about 5mg m of chrysotile asbestos for 5 h. The lengths and diameters of fibres were measured with the transmission electron microscope. In the 90-day period following exposure, there was a progressive increase in the mean length and decrease in the mean diameter of fibres in the lungs. For fibres recovered by broncho-alveolar lavage, both the mean length and diameter decreased with time. These findings indicate that short fibres are cleared preferentially from the lung and that chrysotile fibres progressively separate into individual fibrils. [ABSTRACT FROM PUBLISHER]

The six-circle diffractometer D23 set up in LURE-DCI (bending magnet) at Orsay has been improved since its first presentation in 1987 [M. Bessiere, G. Bessenay, J. Frouin, M. Jouvin, and S. Lefebvre, Nucl. Instrum. Methods A 261, 591 ( 1987)]. We present a new design for the sagittal focusing monochromator, a two-mirror device for harmonics rejection and detectors for adjustments. All these modifications are now operational and can be used together with a crystal analyzer for powder studies. [ABSTRACT FROM AUTHOR]

The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFβ1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5′ RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the –COOH terminal, and an –NH2 terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene. [Copyright &y& Elsevier]

ANT3310 is a novel broad-spectrum diazabicyclooctane serine β-lactamase inhibitor being developed in combination with meropenem (MEM) for the treatment of serious infections in hospitalized patients where carbapenem-resistant Gram-negative pathogens are expected. In this study, we evaluated the in vitro antibacterial activity of MEM in the presence of ANT3310 at 8 µg/mL against global clinical isolates that included Acinetobacter baumannii ( n = 905), carbapenem-resistant Enterobacterales (CRE), carrying either oxacillinase (OXA) ( n = 252) or Klebsiella pneumoniae carbapenemase (KPC) ( n = 180) carbapenemases, and Pseudomonas aeruginosa ( n = 502). MEM was poorly active against A. baumannii, as were MEM-vaborbactam, ceftazidime-avibactam, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and imipenem-relebactam (MIC 90 values of ≥32 µg/mL). On the other hand, MEM-ANT3310 displayed an MIC 90 value of 4 µg/mL, similar to that observed with sulbactam-durlobactam, a drug developed to specifically treat A. baumannii infections. ANT3310 (8 µg/mL) additionally restored the activity of MEM against OXA- and KPC-producing CREs decreasing MEM MIC 90 values from >32 µg/mL to 0.25 and 0.5 µg/mL, respectively. The combination of 8 µg/mL of both MEM and ANT3310 prevented growth of 97.5% of A. baumannii and 100% of OXA- and KPC-positive CREs, with ~90% of P. aeruginosa isolates also displaying MEM MICs ≤8 µg/mL. Furthermore, MEM-ANT3310 was efficacious in both thigh and lung murine infection models with OXA-23 A. baumannii . This study demonstrates the potent in vitro activity of the MEM-ANT3310 combination against both carbapenem-resistant A. baumannii and Enterobacterales clinical isolates, a key differentiator to other β-lactam/β-lactamase combinations., Competing Interests: ANTABIO SAS paid for most of the studies presented in this manuscript. M.Z., P.A., A.L., D.D., S.L., C.S., A.L., J.B., J.C., C.L.,M.E., and M.L. were ANTABIO employees at the time this work was performed.

Pseudomonas aeruginosa is a common pathogen in cystic fibrosis (CF) patients and a major contributor to progressive lung damage. P. aeruginosa elastase (LasB), a key virulence factor, has been identified as a potential target for anti-virulence therapy. Here, we sought to differentiate the P. aeruginosa isolates from early versus established stages of infection in CF patients and to determine if LasB was associated with either stage. The lasB gene was amplified from 255 P. aeruginosa clinical isolates from 70 CF patients from the Toulouse region (France). Nine LasB variants were identified and 69% of the isolates produced detectable levels of LasB activity. Hierarchical clustering using experimental and clinical data distinguished two classes of isolates, designated as 'Early' and 'Established' infection. Multivariate analysis revealed that the isolates from the Early infection class show higher LasB activity, fast growth, tobramycin susceptibility, non-mucoid, pigmented colonies and wild-type lasR genotype. These traits were associated with younger patients with polymicrobial infections and high pFEV 1 . Our findings show a correlation between elevated LasB activity in P. aeruginosa isolates and early-stage infection in CF patients. Hence, it is this patient group, prior to the onset of chronic disease, that may benefit most from novel therapies targeting LasB., (© 2023. Springer Nature Limited.)

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa , a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1β. Furthermore, inhibition of LasB-mediated IL-1β activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

The global dissemination of metallo-β-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE) is a serious public health concern. Specifically, NDM (New Delhi MBL) has been a major cause of carbapenem therapy failures in recent years, particularly as effective treatments for serine-β-lactamase (SBL)-producing Enterobacterales are now commercially available. Since the NDM gene is carried on promiscuous plasmids encoding multiple additional resistance determinants, a large proportion of NDM-CREs are also resistant to many commonly used antibiotics, resulting in limited and suboptimal treatment options. ANT2681 is a specific, competitive inhibitor of MBLs with potent activity against NDM enzymes, progressing to clinical development in combination with meropenem (MEM). Susceptibility studies have been performed with MEM-ANT2681 against 1,687 MBL-positive Enterobacterales , including 1,108 NDM-CRE. The addition of ANT2681 at 8 μg/ml reduced the MEM MIC 50 /MIC 90 from >32/>32 μg/ml to 0.25/8 μg/ml. Moreover, the combination of 8 μg/ml of both MEM and ANT2681 inhibited 74.9% of the Verona integron-encoded MBL (VIM)-positive and 85.7% of the imipenem hydrolyzing β-lactamase (IMP)-positive Enterobacterales tested. The antibacterial activity of MEM-ANT2681 against NDM-CRE compared very favorably to that of cefiderocol (FDC) and cefepime (FEP)-taniborbactam, which displayed MIC 90 values of 8 μg/ml and 32 μg/ml, respectively, whereas aztreonam-avibactam (ATM-AVI) had a MIC 90 of 0.5 μg/ml. Particularly striking was the activity of MEM-ANT2681 against NDM-positive Escherichia coli (MIC 90 1 μg/ml), in contrast to ATM-AVI (MIC 90 4 μg/ml), FDC (MIC 90 >32 μg/ml), and FEP-taniborbactam (MIC 90 >32 μg/ml), which were less effective due to the high incidence of resistant PBP3-insertion mutants. MEM-ANT2681 offers a potential new therapeutic option to treat serious infections caused by NDM-CRE., (Copyright © 2021 Zalacain et al.)

Novel therapies are required to treat chronic bacterial infections in cystic fibrosis (CF) sufferers. The most common pathogen responsible for these infections is Pseudomonas aeruginosa , which persists within the lungs of CF sufferers despite intensive antibiotic treatment. P. aeruginosa elastase (also known as LasB or pseudolysin) is a key virulence determinant that contributes to the pathogenesis and persistence of P. aeruginosa infections in CF patients. The crucial role of LasB in pseudomonal virulence makes it a good target for the development of an adjuvant drug for CF treatment. Herein we discuss the discovery of a new series of LasB inhibitors by virtual screening and computer assisted drug design (CADD) and their optimization leading to compounds 29 and 39 ( K i = 0.16 μM and 0.12 μM, respectively)., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310 , which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

The clinical effectiveness of the important β-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-β-lactamase (MBL) enzymes. To address this resistance issue, multiple β-lactam/β-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem.

Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are increasingly prevalent and have become a major worldwide threat to human health. Carbapenem resistance is driven primarily by the acquisition of β-lactamase enzymes, which are able to degrade carbapenem antibiotics (hence termed carbapenemases) and result in high levels of resistance and treatment failure. Clinically relevant carbapenemases include both serine β-lactamases (SBLs; e.g., KPC-2 and OXA-48) and metallo-β-lactamases (MBLs), such as NDM-1. MBL-producing strains are endemic within the community in many Asian countries, have successfully spread worldwide, and account for many significant CRE outbreaks. Recently approved combinations of β-lactam antibiotics with β-lactamase inhibitors are active only against SBL-producing pathogens. Therefore, new drugs that specifically target MBLs and which restore carbapenem efficacy against MBL-producing CRE pathogens are urgently needed. Here we report the discovery of a novel MBL inhibitor, ANT431, that can potentiate the activity of meropenem (MEM) against a broad range of MBL-producing CRE and restore its efficacy against an Escherichia coli NDM-1-producing strain in a murine thigh infection model. This is a strong starting point for a chemistry lead optimization program that could deliver a first-in-class MBL inhibitor-carbapenem combination. This would complement the existing weaponry against CRE and address an important and growing unmet medical need., (Copyright © 2018 American Society for Microbiology.)

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells., (© 2011 John Wiley & Sons A/S.)

Toxin-antitoxin systems, as found in bacterial plasmids and their host chromosomes, play a role in the maintenance of genetic information, as well as in the response to stress. We describe the basic biology of the parD/kiskid toxin-antitoxin system of Escherichia coli plasmid R1, with an emphasis on regulation, toxin activity, potential applications in biotechnology and its relationships with related toxin-antitoxin systems. Special reference is given to the ccd toxin-antitoxin system of plasmid F because its toxin shares structural homology with the toxin of the parD system. Inter-relations with related toxin-antitoxin systems present in the E. coli chromosome, such as the parD homologues chpA/mazEF and chpB and the relBE system, are also reviewed. The combined structural and functional information that is now available on all these systems, as well as the ongoing controversy regarding the role of the chromosomal toxin-antitoxin loci, have made this review especially timely.

Generational coexistence in structured environments raises the possibility of a competition between ancestors and descendents. This type of kin competition, and in particular, the possibility that descendents might actively repress the ancestor's dominance, has been rarely considered in microbial evolutionary ecology. The recent discovery of the phenomenon of stationary-phase contact-dependent inhibition of bacterial ancestor cells by late descendents provides a new theoretical perspective to analyze intrapopulational evolutionary changes. The ancestor's inhibition effect might accelerate such changes, particularly when the descendents have acquired small adaptive advantages that are insufficient to rapidly displace the well-settled ancestors in a complex niche. Besides this effect of triggering selection of small genetic differences, the opportunities for intergenerational coexistence in bacteria, where ancestor's inhibition might occur, are reviewed in this work. A theoretical analysis is provided about the explanatory possibilities of the ancestor's inhibition effect in the controversies about intraspecific (in a large sense, including intrapopulational) genetic diversification, and the discontinuities observed in such processes, giving rise to the emergence of individualities and therefore differential units of selection.

Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.

In the course of liquid culture, serial passage experiments with Escherichia coli K-12 bearing a mutator gene deletion (DeltamutS) we observed the evolution of strains that appeared to kill or inhibit the growth of the bacteria from where they were derived, their ancestors. We demonstrate that this inhibition occurs after the cells stop growing and requires physical contact between the evolved and ancestral bacteria. Thereby, it is referred to as stationary phase contact-dependent inhibition (SCDI). The evolution of this antagonistic relationship is not anticipated from existing theory and experiments of competition in mass (liquid) culture. Nevertheless, it occurred in the same way (parallel evolution) in the eight independent serial transfer cultures, through different single base substitutions in a gene in the glycogen synthesis pathway, glgC. We demonstrate that the observed mutations in glgC, which codes for ADP-glucose pyrophosphorylase, are responsible for both the ability of the evolved bacteria to inhibit or kill their ancestors and their immunity to that inhibition or killing. We present evidence that without additional evolution, mutator genes, or known mutations in glgC, other strains of E. coli K-12 are also capable of SCDI or sensitive to this inhibition. We interpret this, in part, as support for the generality of SCDI and also as suggesting that the glgC mutations responsible for the SCDI, which evolved in our experiments, may suppress the action of one or more genes responsible for the sensitivity of E. coli to SCDI. Using numerical solutions to a mathematical model and in vitro experiments, we explore the population dynamics of SCDI and postulate the conditions responsible for its evolution in mass culture. We conclude with a brief discussion of the potential ecological significance of SCDI and its possible utility for the development of antimicrobial agents, which unlike existing antibiotics, can kill or inhibit the growth of bacteria that are not growing.

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed., ((c) 2007 Wiley-Liss, Inc.)

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5' to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the alpha-cardiac actin locus.

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent cleavage occurred at target sequences situated either in single- or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.

The toxin-antitoxin system parD (kis kid) of plasmid R1 is coregulated by the coordinated action of its two gene products. Here we describe the isolation and the in vivo characterization of three single-amino-acid changes in the Kid toxin, G4E, C74Y, and E91K, that affect the coregulatory activity but preserve the toxicity of the protein.

The molecular mechanisms that establish and maintain transcriptional differences between cardiomyocytes in the left and right ventricular chambers are unkown. We have previously analysed a myosin light chain 3f transgene containing an nlacZ reporter gene, which is transcribed in left but not right ventricular cardiomyocytes. In this report we examine the mechanisms involved in maintaining regionalised transgene expression. Primary cardiomyocytes prepared from left and right ventricular walls of transgenic mice were found to maintain transgene expression status in culture. However, similar cultures prepared from nontransgenic mice or rats show uniform expression after transient transfection of Mlc3f constructs, suggesting that the mechanism responsible for differential expression of the transgene between left and right ventricular cells does not operate on transiently introduced molecules. These data suggest that developmental cell history determines transgene expression status. Maintenance of transgene expression status is regulated by a cell-autonomous mechanism that is independent of DNA methylation, trichostatin A-sensitive histone deacetylation and miss-expression of transcription factors that are expressed in the left or right ventricles of the embryonic heart. Parallels between Mlc3f transgene repression in right ventricular cardiomyocytes and polycomb-mediated silencing in Drosophila suggest that Mlc3f regulatory sequences included on the transgene may contain a cellular memory module that is switched into an on or off state during early cardiogenesis. Epigenetic mechanisms may therefore be involved in maintaining patterning of the mammalian myocardium.

Kid and Kis are, respectively, the toxin and antitoxin encoded by the parD operon of plasmid R1. The recently solved crystal structure of Kid has revealed that this protein closely resembles the CcdB toxin of plasmid F. In CcdB, the residues involved in toxicity are located at the carboxy-terminal end of the protein. However, an analogous information on the Kid toxin was not available. Here, we have characterized a collection of non-toxic mutants of the Kid protein and identified the residues that affected the toxicity but not the co-regulatory activity of Kid. These are located in two discrete regions of the protein, at the amino and carboxy-terminal ends. Particularly, residues E18 and R85, that are conserved in the Escherichia coli ChpAK and RelE toxins, are affected by amino-acid changes that alter neither the overall structure of the protein nor its state of association, as shown by CD and sedimentation equilibrium analyses. However, thermal denaturation and intrinsic tryptophan fluorescence emission data point to subtle local changes at the N-terminal end of the protein. The implications of these results in the current model on the structure and function of Kid-related bacterial toxins are discussed.

Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30 degrees C, but not at 42 degrees C. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB-Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth.