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1.
Methods Mol Biol ; 1333: 43-52, 2016.
Article in English | MEDLINE | ID: mdl-26468098

ABSTRACT

Monitoring persister cells can be extremely difficult due to their transient and stochastic nature, their low abundance, and their resemblance to Viable But Non-Culturable Cells (VBNCs). To date, the predominant method consists of determining the survival rate of a bacterial population after antibiotic treatment as a function of time or antibiotic concentration. Unfortunately, this method is limited, as it shows high levels of dispersion of the data around the mean, making interpretation difficult. Furthermore, additional reproducibility problems arise from the lack of a standard method, different research groups using different protocols. Here, we describe a standard and optimized method for monitoring E. coli persister cells at the population level allowing for maximal reproducibility.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Drug Resistance, Bacterial/genetics , Escherichia coli/drug effects , Colony Count, Microbial , Dose-Response Relationship, Drug , Drug Resistance, Bacterial/drug effects , Humans , Microbial Sensitivity Tests
2.
Clin Microbiol Infect ; 19(5): E222-9, 2013 May.
Article in English | MEDLINE | ID: mdl-23464795

ABSTRACT

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.


Subject(s)
Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/genetics , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Carrier Proteins/immunology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Genetic Variation , Global Health , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Analysis, DNA , Streptococcal Infections/microbiology , Streptococcus pyogenes/immunology , Streptococcus pyogenes/isolation & purification
3.
Clin Microbiol Infect ; 17(6): 907-14, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20977542

ABSTRACT

A prospective cohort study of preschool healthy children (3-6 years old) from two distinct socio-economic settings in the Brussels area, Belgium, was conducted during the years 2006-2008. The objectives were to evaluate nasopharyngeal colonization by Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis and Haemophilus influenzae at the time of PCV7 vaccine introduction and to assess the socio-economic level impact on flora composition and antibiotic resistance. Three hundred and thirty-three children were included and a total of 830 nasopharyngeal samples were collected together with epidemiological data. Pneumococcal serotypes and antibiotic resistance profiles were determined. Risk factors for carriage and bacterial associations were analysed by multivariate logistic regression. Carriage rates were high for all pathogens. Fifty per cent of the children were colonized at least once with S. aureus, 69% with S. pneumoniae, 67% with M. catarrhalis and 83% with H. influenzae. PCV7 uptake was higher among children from a higher socio-economic setting and S. pneumoniae serotypes varied accordingly. Children from lower socio-economic schools were more likely to carry M. catarrhalis, S. aureus and antibiotic-resistant S. pneumoniae, including a high proportion of non-typeable pneumococcal strains. Positive associations between S. pneumoniae and H. Influenza, between H. influenzae and M. catarrhalis and between H. influenzae and S. aureus were detected. Our study indicates that nasopharynx flora composition is influenced not only by age but also by socio-economic settings. A child's nasopharynx might represent a unique dynamic environment modulated by intricate interactions between bacterial species, host immune system and PCV7 immunization.


Subject(s)
Bacterial Infections/epidemiology , Carrier State/epidemiology , Nasopharynx/microbiology , Anti-Bacterial Agents/pharmacology , Bacterial Infections/microbiology , Bacterial Typing Techniques , Belgium/epidemiology , Carrier State/microbiology , Child , Child, Preschool , Cohort Studies , Drug Resistance, Bacterial , Female , Haemophilus influenzae/drug effects , Haemophilus influenzae/isolation & purification , Humans , Male , Microbial Sensitivity Tests , Moraxella catarrhalis/drug effects , Moraxella catarrhalis/isolation & purification , Prevalence , Prospective Studies , Risk Factors , Serotyping , Socioeconomic Factors , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purification , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/isolation & purification
4.
Mol Microbiol ; 41(1): 73-82, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11454201

ABSTRACT

The ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity. Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear. The results presented here indicate that, although CcdA is the DNA-binding element of the CcdA-CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator-promoter region. Using electrophoretic mobility shift assays, we show that a (CcdA)2-(CcdB)2 complex binds DNA. The addition of extra CcdB to that protein-DNA complex completely abolishes DNA retardation. Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon. When the level of CcdA is superior or equal to that of CcdB, repression results. In contrast, derepression occurs when CcdB is in excess of CcdA. By ensuring an antidote-toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid-containing bacteria.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Plasmids/genetics , Transcription, Genetic , Bacterial Proteins/genetics , Bacterial Toxins/genetics , DNA, Bacterial/metabolism , Electrophoresis, Polyacrylamide Gel , Mutation , Operator Regions, Genetic/genetics , Operator Regions, Genetic/physiology , Operon/genetics , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology
5.
Proc Natl Acad Sci U S A ; 96(11): 6064-71, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10339542

ABSTRACT

Lon protein of Escherichia coli is an ATP-dependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a lon deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a Deltalon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATP-dependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins.


Subject(s)
Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/metabolism , Protease La , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acid Substitution , Arabinose/metabolism , Bacterial Proteins/metabolism , Binding Sites , Cell Division , Consensus Sequence , Escherichia coli/genetics , Escherichia coli/radiation effects , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Kinetics , Models, Chemical , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Substrate Specificity , Ultraviolet Rays
6.
J Mol Biol ; 285(4): 1667-77, 1999 Jan 29.
Article in English | MEDLINE | ID: mdl-9917404

ABSTRACT

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix. In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix. The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Escherichia coli/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Crystallography, X-Ray , DNA Gyrase , DNA, Bacterial/metabolism , Dimerization , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrogen Bonding , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Mutation , Plasmids/genetics , Protein Conformation , Protein Structure, Secondary , Topoisomerase II Inhibitors
7.
Trends Microbiol ; 6(7): 269-75, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9717215

ABSTRACT

DNA gyrase is an essential topoisomerase that is found in all bacteria and is the target of potent antibiotics, such as the quinolones. By creating DNA lesions and inducing the bacterial SOS response, these drugs are not only highly cytotoxic but also mutagenic. Discovery and analysis of natural molecules with anti-gyrase activities, such as the CcdB or microcin B17 proteins, hold promise for understanding further topoisomerase reactions and for the design of new antibiotics.


Subject(s)
Bacteria/drug effects , Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Topoisomerase II Inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology
8.
J Biol Chem ; 273(1): 524-7, 1998 Jan 02.
Article in English | MEDLINE | ID: mdl-9417111

ABSTRACT

The bacteriophage T4 PinA protein inhibited degradation of [3H]alpha-methyl casein by purified Lon protease from Escherichia coli, but inhibition was noncompetitive with respect to casein. PinA did not inhibit cleavage of the fluorogenic peptide, N-glutaryl-alanylalanylphenylalanyl-3-methoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic peptides by Lon. Thus, PinA does not block the proteolytic active site or the allosteric protein-binding site on Lon. Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activated ATPase activity was usually 80-95%. Inhibition was noncompetitive with respect to ATP. PinA did not block activation of peptide cleavage by nonhydrolyzable analogs of ATP. These data suggest that PinA does not bind at the ATPase active site of Lon and does not interfere with nucleotide binding to the enzyme. PinA inhibited cleavage of the 72-amino acid protein, CcdA, degradation of which requires ATP hydrolysis, but did not inhibit cleavage of the carboxyl-terminal 41-amino acid fragment of CcdA, degradation of which does not require ATP hydrolysis. PinA thus appears to interact at a novel regulatory or enzymatic site involved in the coupling between ATP hydrolysis and proteolysis, possibly blocking the protein unfolding or remodeling step essential for degradation of high molecular weight protein substrates by Lon.


Subject(s)
Adenosine Triphosphate/metabolism , Escherichia coli Proteins , Heat-Shock Proteins/metabolism , Protease Inhibitors/metabolism , Protease La , Serine Endopeptidases/metabolism , Viral Proteins/metabolism , ATP-Dependent Proteases , Bacteriophage T4/metabolism , Hydrolysis , Protein Denaturation
9.
J Biol Chem ; 271(44): 27730-8, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8910366

ABSTRACT

CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km >/=200 microM), and the peptide bond turnover number was approximately 10 min-1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost alpha-helical structure at elevated temperatures (Tm approximately 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.


Subject(s)
Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/metabolism , Protease La , Protein Structure, Secondary , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Bacterial Toxins/chemistry , Bacterial Toxins/isolation & purification , Bacterial Toxins/metabolism , Binding Sites , Chromatography, Gel , Chromatography, High Pressure Liquid , Circular Dichroism , Escherichia coli/genetics , F Factor , Heat-Shock Proteins/isolation & purification , Kinetics , Macromolecular Substances , Molecular Sequence Data , Operon , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine Endopeptidases/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
Mol Microbiol ; 15(6): 1031-7, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7623659

ABSTRACT

The ccd locus of the F plasmid codes for two gene products, CcdA and CcdB, which contribute to the plasmid's high stability by post-segregational killing of plasmid-free bacteria. Like the quinolones, the CcdB protein is a poison of the DNA-topoisomerase II complexes, while CcdA acts as an antidote against CcdB. In addition to these poison-antipoison properties, the CcdA and CcdB proteins act together at transcription level to repress their own synthesis. In this work, we have isolated, in vivo, and characterized several non-killer CcdB mutants. All missense mutations which inactivate CcdB killer activity are located in the region coding for the last three C-terminal residues. However, the resulting mutant CcdB proteins retain their autoregulatory properties. We conclude that the last three C-terminal residues of CcdB play a key role in poisoning but are not involved in repressor formation.


Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins/genetics , Escherichia coli/genetics , F Factor/genetics , Mutation/physiology , Bacterial Proteins/biosynthesis , Bacterial Toxins/biosynthesis , Base Sequence , DNA Mutational Analysis , DNA, Bacterial/metabolism , Molecular Sequence Data , Operator Regions, Genetic/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Suppression, Genetic/genetics
11.
Mol Gen Genet ; 244(5): 530-8, 1994 Sep 01.
Article in English | MEDLINE | ID: mdl-8078480

ABSTRACT

The ccd operon of the F plasmid contributes to the high stability of the episome by postsegregational killing of plasmid-free bacteria. It contains two genes, ccdA and ccdB, which are negatively autoregulated at the level of transcription, probably by a complex comprising the two gene products. Using the bacterial gyrA462 CcdB resistance mutation and a Pccd-lacZ transcriptional fusion, we have obtained evidence that the CcdB protein by itself has no regulatory activity or operator DNA-binding affinity and needs CcdA in order to effect transcriptional control. The ccd killing mechanism is based on the poison-antidote principle. The CcdB protein is cytotoxic, poisoning DNA-gyrase complexes, while CcdA antagonizes this activity. In order to define functional domains of the CcdA antidote involved in the anti-killer effect, autoregulation or both, we introduced several missense or amber mutations into the CcdA protein by directed mutagenesis. We report on missense CcdA proteins that have lost their autoregulatory properties but are still able to antagonize the lethal activity of CcdB. We show that the five carboxy-terminal amino acid residues of the antidote protein are not required for the antidote effect or for autoregulation. Several missense CcdA polypeptides were generated by suppression of nonsense codons. Two substitutions lead to CcdB-promoted killing: glutamine 33-->cysteine and glutamine 33-->phenylalanine.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Bacterial Toxins/antagonists & inhibitors , Escherichia coli/genetics , F Factor , Gene Expression Regulation, Bacterial , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Bacteriolysis , Base Sequence , Cloning, Molecular , Cytotoxins/antagonists & inhibitors , Cytotoxins/metabolism , DNA Damage , DNA Mutational Analysis , DNA Primers , DNA Topoisomerases, Type II/metabolism , DNA, Bacterial/genetics , Escherichia coli/metabolism , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Operon , Point Mutation , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Suppression, Genetic , beta-Galactosidase/metabolism
12.
Mol Microbiol ; 11(6): 1151-7, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8022284

ABSTRACT

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein. In this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Heat-Shock Proteins/metabolism , Plasmids/genetics , Protease La , Serine Endopeptidases/metabolism , ATP-Dependent Proteases
13.
J Mol Biol ; 234(3): 534-41, 1993 Dec 05.
Article in English | MEDLINE | ID: mdl-8254658

ABSTRACT

DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation. Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F. When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin. In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex. The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase. The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics. However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present. We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa. Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Cytotoxins/metabolism , DNA Topoisomerases, Type II/metabolism , Escherichia coli/metabolism , F Factor , Adenosine Triphosphate/metabolism , Bacterial Proteins/isolation & purification , Bacterial Toxins/isolation & purification , Base Sequence , DNA Primers , DNA Topoisomerases, Type II/isolation & purification , Escherichia coli/genetics , Genes, Bacterial , Kinetics , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Restriction Mapping , Topoisomerase II Inhibitors
14.
J Mol Biol ; 231(2): 513-5, 1993 May 20.
Article in English | MEDLINE | ID: mdl-8389886

ABSTRACT

Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate. The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A. Both crystal forms diffract to 2.6 A resolution. Structure determination by multiple isomorphous replacement is under way.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Escherichia coli/chemistry , Bacterial Proteins/isolation & purification , Bacterial Toxins/isolation & purification , Circular Dichroism , Crystallization , F Factor , Topoisomerase I Inhibitors , X-Ray Diffraction
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