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. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.

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. pylori SS1 or SS1-rdxA(-), an SS1-derived mutant in rdxA. Twelve weeks after inoculation, the mice were treated for 7 days with a control solution or with the mouse equivalent of a human dose of metronidazole 1 g od. Three weeks after completion of treatment, the animals were killed and mutations in the target lacI gene assessed by a transgenic mutagenesis assay system. There was no increase in lacI mutations in cells harvested from mice infected with H. pylori and/or exposed to metronidazole. These data suggest that short-term infection with H. pylori and exposure to metronidazole does not enhance the mutation frequency in the gastric cells of mice. Whether chronic infection and/or repeated exposure to metronidazole or other nitroaromatic compounds causes genetic damage to gastric epithelial cells remains to be determined.

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. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

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. - treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.

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. The most striking effect of SL3261*pMalE administration was the activation and stimulation of new MalE peptide-specific T-cell responses that were silent after administration of purified Ag with adjuvant. Ag presentation assays performed with MalE-specific T-cell hybridomas showed that infection of Ag-presenting cells by this intracellular attenuated bacterium did not affect the processing and presentation of the different MalE peptides by major histocompatibility complex (MHC) class II molecules and therefore did not account for immunodominance modulation. Thus, immunodominance of the T-cell response to microorganisms is governed not only by the frequency of the available T-cell repertoire or the processing steps in Ag-presenting cells that lead to MHC presentation but also by other parameters probably related to the infectious process and to the bacterial products. Our results indicate that, upon infection by a microorganism, the specificity of the T-cell response induced against its Ags can be much more effective than with purified Ags and that it cannot completely be mimicked by purified Ags administered with adjuvant.

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. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

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. We show that: (i) the substitution of the pair of strands beta13-beta14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta13-beta14 the portion of the E. coli LamB protein comprising strands beta13 to beta16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.

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. Electron microscopy further demonstrated that the fusion protein could also bind to LamB at the surface of intact cells. This interaction prevented lambda adsorption but affected only partially maltose uptake.

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. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.

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. In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6. While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.

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. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.

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.e.--the C3 epitope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145)--at the tip of the most distal and largest surface exposed region of LamB (after residues 386, into loop L9). We also used two previously constructed LamB hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two LamB proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site). The LamB hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S. typhimurium, SL3261. In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained. For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the LamB hybrid corresponding to the simultaneous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hepatitis B antibodies.

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. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems.

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. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.

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. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed.

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. Mol. Biol., 1994, Vol. 238, pp. 319-332]. It was found that R7000 induced a broad spectrum of low frequency replication blocks rather than particular hot spots in a limited number of particular targets. Most of these replication blocks were observed at G nucleotides, and most of G nucleotides present in the DNA sequence, if not all, constituted a possible target for the chemical attack of the compound. In addition, a large part of replication blocks observed at A, C or T could also reflect a replication block at the 3' or 5' nucleotide flanking a guanosine-DNA adduct. Only a very small number of replication blocks could be observed at A, C or T nucleotides non-adjacent to a G. These results show that, guanosine-DNA adducts are the main DNA lesions that block replication induced by R7000 in E. coli and suggests a strong reactivity of the genotoxic species generated in vivo by R7000 with the G nucleotidic targets. From 26 R7000-induced mutations previously mapped in this region [E. Touati, E. Krin, P. Quillardet, M. Hofnung, 7-methoxy-2-nitronaphto[2,1-b]furan (R7000)-induced mutation spectrum in the lacI gene of Escherichia coli: influence of SOS mutagenesis, Carcinogenesis, 1996, Vol. 17, pp. 2543-2550.], 22 (85%) occurred at GC base pairs at which termination products were observed. The other mutagenic events involved AT base pairs adjacent to a G nucleotide forming a replication block. Thus all mutagenic events occurred at, or adjacent to, a G nucleotide forming a replication block. Although it could not be excluded that some mutagenic events are due to undetected DNA lesions that do not block replication, these results strongly suggest that guanosine-DNA adducts that block DNA replication are responsive for a large part of the mutagenic events generated by R7000. The powerful capacity of R7000 to form adducts at most of the guanosine residues in a DNA sequence may account for at least part of its very potent genotoxic properties.

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. The present work describe first in vitro studies on the interactions between J and LamB. The J gene of lambda was cloned into a plasmid vector under ptac promoter control and expressed in E. coli. We showed that J could be expressed at high levels (up to 28% of whole cell proteins), in an insoluble form. Anti-J antibodies, induced in rabbits immunized with insoluble J extracts, appeared to specifically neutralize lambda infection. Under defined conditions of extraction, the J protein was obtained in a soluble form. We showed that solubilized J was able to interact with LamB trimers in vitro. Implications for future studies on the interactions between LamB and J are discussed.

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. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super-secondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.

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. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.