Iris Thamm

Structure of In31, a blaIMP-Containing Pseudomonas aeruginosa Integron Phyletically Related to In5, Which Carries an Unusual Array of Gene Cassettes

ABSTRACT The location and environment of the acquiredblaIMP gene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a JapanesePseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate,blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes anaacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.

The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene

A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor.

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

Abstract. Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

The Division and Cell Wall Gene Cluster of Enterococcus Hirae S185

A chromosomal 10355-bp segment of Enterococcus hirae S185 contains nine orfs which occur in the same order as the MraW-, FtsL-, PBP3-, MraY-, MurD-, MurG-, FtsQ-, FtsA- and FtsZ-encoding genes of the division and cell wall clusters of Escherichia coli and Bacillus subtilis. The E. hirae DNA segment lacks the genes which in E. coli encode the ligases Ddl, MurC, MurE and MurF and the integral membrane protein FtsW. The encoded E. hirae and E. coli proteins share 25% to 50% identity except FtsL and FtsQ (approximately = 14% identity).

Saturation of Penicillin-Binding Protein 1 by Beta-Lactam Antibiotics in Growing Cells of Bacillus Licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin‐binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β‐lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.