Genotoxicity of 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000): a case study with some considerations on nitrofurantoin and nifuroxazide.

Two nitrofurans present broad-spectrum antimicrobial properties and some of them are used in human and veterinary medicine. Most of these molecules are mutagens and some of them were reported as carcinogens. Due to its extreme mutagenic potency in bacteria, the nitronaphtho derivative 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000) was used as a tool to analyze the mechanism of the genotoxic action of this family of chemicals.

Palindromic unit-independent transposition of IS1397 in Yersinia pestis.

Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes of Escherichia coli and several other enterobacteria. In the latter, IS1397, an E. coli insertion sequence specific to PUs, transposes into PUs with sequences close to the E.

Comparison of kinetics of induction of DNA adducts and gene mutations by a nitrofuran compound, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in the caecum and small intestine of Big Blue mice.

In previous experiments, i.p. injection of the 5 nitronaphthofuran derivative 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) to lacI transgenic Big Blue mice led to an increase in the mutant frequency (MF), especially in the caecum and the small intestine.

Transposition of IS1397 in the family Enterobacteriaceae and first characterization of ISKpn1, a new insertion sequence associated with Klebsiella pneumoniae palindromic units.

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species.

Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein.

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope.

Short-term infection with Helicobacter pylori and 1 week exposure to metronidazole does not enhance gastric mutation frequency in transgenic mice.

The aim of this study was to determine whether exposure of Helicobacter pylori-infected mice to metronidazole resulted in the delivery of mutagenic compounds to the gastric epithelium via the oxygen-insensitive NADPH nitroreductase (RdxA) of H. pylori. C57BL/6 transgenic mice containing the lambda/lacI transgene were inoculated with peptone trypsin broth, H.

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2, 1-b]furan (R7000), in lacI transgenic mice.

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p.

Extending the CD4(+) T-cell epitope specificity of the Th1 immune response to an antigen using a Salmonella enterica serovar typhimurium delivery vehicle.

We analyzed the CD4 T-cell immunodominance of the response to a model antigen (Ag), MalE, when delivered by an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261*pMalE). Compared to purified MalE Ag administered with adjuvant, the mapping of the peptide-specific proliferative responses showed qualitative differences when we used the Salmonella vehicle. We observed the disappearance of one out of eight MalE peptides' T-cell reactivity upon SL3261*pMalE immunization, but this phenomenon was probably due to a low level of T-cell priming, since it could be overcome by further immunization.

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane.

In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin.

LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro.

Short palindromic repetitive DNA elements in enterobacteria: a survey.

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats.

The C-terminal portion of the tail fiber protein of bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, its cell-surface receptor. We fused C-terminal portions of J, the tail fiber protein of lambda, to maltose-binding protein. Solid-phase binding assays demonstrated that a purified fusion protein comprising only the last 249 residues of J could bind to LamB trimers and inhibited recognition by anti-LamB antibodies.

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements.

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella.

In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding.

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro.

Structure-function study of MalF protein by random mutagenesis.

MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK.

Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites of a carrier protein. By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number of different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.

Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters.

ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database.

A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake.

The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues.

DNA damage induced in vivo by 7-methoxy-2-nitronaphtho[2,1-b]-furan (R7000) in the lacI gene of Escherichia coli.

DNA adducts that block replication, induced in vivo by the 5-nitrofuran derivative R7000 (7-methoxy-2-nitronaphtho[2, 1-b]-furan) were mapped, at nucleotide resolution, in a region of the lacI gene of Escherichia coli, using a reiterative primer extension assay [D. Chandrasekhar, B. Van Houten, High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene: inefficient repair of the non-transcribed strand correlates with high mutation frequency, J.

Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor.

Bacteriophage lambda adsorbs to its Escherichia coli K12 host by interacting with a specific cell surface receptor, the outer membrane protein LamB. Previous genetic analyses led us to define a set of residues at the surface of LamB, which belong to the lambda receptor site. Further genetic studies indicated that the C-terminal portion of J, the tail fibre protein of lambda, was directly involved in the recognition of the receptor site.

Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein.

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alphaI/betaB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alphabeta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies.

Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli.

The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation.

Immunodominance does not result from peptide competition for MHC class II presentation.

Competition for binding to MHC class II molecules between processed peptides derived from a single protein Ag is considered an important parameter leading to the presentation of a limited set of peptides by APCs. We tested the relevance of this competition process in a model Ag, the MalE protein, by deleting T cell epitopes or by introducing a competitor T cell peptide. We identified in DBA/1 (I-Aq) mice six immunodominant T cell determinants in the MalE sequence, 89-95, 116-123, 198-205, 211-219, 274-281, and 335-341.