Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrasemediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn2l and Tn5O86. Several characteristics suggest that this element is a transposon, which we call TnSO9O. TnS090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins.

Among several observations of greatly increased levels of chromosomal dihydrofolate reductase as a cause of resistance to high concentrations of the antifolate drug trimethoprim, in clinically isolated bacteria, one is described here of a strain of Escherichia coli overproducing dihydrofolate reductase several hundredfold. The chromosomally located resistance gene of this strain was isolated, inserted into a plasmid vector, and analyzed for its nucleotide sequence. The structural gene for the overproduced dihydrofolate reductase was found to be identical to that of E. coli K 12, with nine exceptions, of which seven resulted in synonymous codon usage. Two transversions resulted in a substitution of Gly for Trp at amino acid position 30, and of Gln for Glu at position 154. Six of the nine base changes resulted in codons more frequently used. The Gly substitution which leads to a less commonly used codon, was thought to relate to the observed threefold increase in Ki for trimethoprim. Furthermore, a C+T transition was found in the -35 region of the promoter, increasing its homology with the E. coEi consensus promoter sequence. In the ribosome-binding area of the resistant strain, finally, seven base changes were observed, two of which resulted in a five-base sequence of complementarity with the 3'-end of ribosomal 16s RNA. The distance between the -10 site of the promoter and the start codon for translation was finally increased one base pair by the insertion of an A at position + 9 in the resistant strain. These genetic changes towards more efficient transcriptional and translational start sequences and towards increased mRNA expressivity are interpreted to reflect an evolutionary adaptation to the presence of antifolates.

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.

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EMBL accession no. X51522 P4 is a satellite bacteriophage of Escherichia coli that requires the products of the late genes of phages of the P2 family for lytic multiplication (1). We present here the complete DNA sequence of P4.