Elegant studies by C. Hill’s group on the effect of mutations in 6 of the genes encoding PBPs (including lmo1438) on the susceptibility of L. monocytogenes to β-lactams, revealed that lmo0441 and lmo2229 (PBP4) contribute to the β-lactam
resistance of L. monocytogenes, but inactivation of lmo1438 did not result MK-8776 clinical trial in obvious changes to either the sensitivity to β-lactams or the cell morphology [8]. Taking into account the seemingly contradictory nature of the aforementioned reports and the fact that the gene encoding PBP3 has yet to be directly identified, plus the absence of reports regarding the physiological function of this protein, our study focused on gene lmo1438 (potentially encoding PBP3). Here we describe the use of the lactococcal nisin-controlled expression (NICE) system [10] for the overexpression of this gene. This strategy was chosen because in a recently described analysis, mutational inactivation of lmo1438 had no obvious physiological effect [8]. In the present study, it has been directly demonstrated that lmo1438 encodes L. monocytogenes PBP3. Overexpression of this protein, which was accompanied by a slight increase in PBP4 expression, Selleck S3I-201 resulted in growth
retardation, shortening of cells in the stationary phase of growth and minor changes in the susceptibility of L. monocytogenes to β-lactams. The observed changes in cell morphology indicate the involvement SIS3 of PBP3 in cell division. These novel data on the overexpression of gene lmo1438 provide a more comprehensive view of the physiological function of PBP3 and its significance in the susceptibility of L. monocytogenes to β-lactams. These findings also further our understanding of the mechanisms of L. monocytogenes susceptibility to β-lactams, which is of direct relevance to its antibiotic resistance, the use of antibiotic therapy to treat listeriosis, as well as the ability of this bacterium to form biofilms [2, 11]. Results and discussion Construction of plasmid pAKB carrying the nisin-controlled expression (NICE) system DAPT mouse and its application in
L. monocytogenes Given the contradictory reports on the significance of PBP3 in the susceptibility of L. monocytogenes to β-lactam antibiotics, it was decided to study the effects of overexpression of L. monocytogenes gene lmo1438. The lactococcal NICE system [10] was chosen for overexpression studies since it has previously been successfully used in a number of gram-positive genera, including L. monocytogenes [12–15]. This system consists of a two-component signal transduction system NisRK, which senses the presence of nisin and induces transcription from the promoter Pnis. Recently developed strategies for using the NICE system either place the nisRK genes on the host chromosome, which allows the use of a single-plasmid system with a nisA promoter [13], or place both nisRK and the nisA promoter on one plasmid [16]. The first strategy was successfully used in L. monocytogenes by Cotter et al.