Marie-Françoise Prère

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.

Phage-Antibiotic Synergy (PAS): β-Lactam and Quinolone Antibiotics Stimulate Virulent Phage Growth

Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cells production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strains production of the phage ΦMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with β-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

Transposase-induced excision and circularization of the bacterial insertion sequence IS911

We have investigated the role of three IS911‐specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by −1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB‐mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site‐specific intramolecular transposition event. We present a model which explains both intra‐ and intermolecular transposition events in terms of a single reaction mechanism of the ‘cut and paste’ type.

Impact of Three Ampicillin Dosage Regimens on Selection of Ampicillin Resistance in Enterobacteriaceae and Excretion of blaTEM Genes in Swine Feces

ABSTRACT The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of blaTEM genes, which code for the most frequently produced β-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and blaTEM gene quantities were below 107 copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 μg/ml). In the control group, blaTEM gene quantities fluctuated between 104 and 106 copies/g of feces, whereas they fluctuated between 106 to 108 and 107 to 109 copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, blaTEM gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal blaTEM gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment.

Relatedness of Escherichia coli strains with different susceptibility phenotypes isolated from swine feces during ampicillin treatment.

ABSTRACT The aim of this study was to examine the dynamics of the development of resistance in fecal Escherichia coli populations during treatment with ampicillin for 7 days in pigs. Before treatment, only 6% of the isolates were ampicillin resistant, whereas more than 90% of the isolates were resistant after days 4 and 7 of treatment. Ampicillin-resistant E. coli isolates were mainly multiresistant, and 53% of the isolates from the treated pigs had one phenotype that included resistance to six antibiotics (ampicillin, chloramphenicol, sulfonamides, tetracycline, trimethoprim, and streptomycin) at day 7. Determination of the frequency of the four phylogenetic groups showed that there was a shift in the E. coli population in ampicillin-treated pigs; before treatment 75% of the isolates belonged to phylogroup B1, whereas at day 7 85% of the isolates belonged to phylogroup A. Pulsed-field gel electrophoresis (PFGE) typing revealed that ampicillin treatment selected ampicillin-resistant isolates with genotypes which were present before treatment. Comparison of antimicrobial phenotypes and PFGE genotypes showed that resistance traits were disseminated by vertical transmission through defined strains. One PFGE genotype, associated with the six-antibiotic-resistant phenotype and including a specific combination of resistance determinants, was predominant among the ampicillin-resistant strains before treatment and during treatment. These data indicate that ampicillin administration selected various ampicillin-resistant isolates that were present in the digestive tract before any treatment and that E. coli isolates belonging to one specific PFGE genotype encoding resistance to six antibiotics became the predominant strains as soon as ampicillin was present in the digestive tract.

Apical Loop-Internal Loop RNA Pseudoknots A NEW TYPE OF STIMULATOR OF-1 TRANSLATIONAL FRAMESHIFTING IN BACTERIA

Nearly all members of a widespread family of bacterial transposable elements related to insertion sequence 3 (IS3), therefore called the IS3 family, very likely use programmed -1 ribosomal frameshifting to produce their transposase, a protein required for mobility. Comparative analysis of the potential frameshift signals in this family suggested that most of the insertion sequences from the IS51 group contain in their mRNA an elaborate pseudoknot that could act as a recoding stimulator. It results from a specific intramolecular interaction between an apical loop and an internal loop from two stem-loop structures. Directed mutagenesis, chemical probing, and gel mobility assays of the frameshift region of one element from the IS51 group, IS3411, provided clear evidences of the existence of the predicted structure. Modeling was used to generate a three-dimensional molecular representation of the apical loop-internal loop complex. We could demonstrate that mutations affecting the stability of the structure reduce both frameshifting and transposition, thus establishing the biological importance of this new type of RNA structure for the control of transposition level.

Translational Defects in a Mutant Deficient in YajL, the Bacterial Homolog of the Parkinsonism-Associated Protein DJ-1

We report here that YajL is associated with ribosomes and interacts with many ribosomal proteins and that a yajL mutant of Escherichia coli displays decreased translation accuracy, as well as increased dissociation of 70S ribosomes into 50S and 30S subunits after oxidative stress.