monocytogenes pAKB-lmo1438 compared with L. monocytogenes pAKB, when both were cultured in the presence of nisin, indicated that this phenomenon is a consequence of PBP3 overexpression. Figure 2 Pattern of PBPs in L. monocytogenes BI 6727 molecular weight strain overexpressing lmo1438. Membrane proteins (200 μg of total protein) of L. monocytogenes pAKB (lane 1) and L. monocytogenes pAKB-lmo1438 (lane 2) were incubated with [3H]benzylpenicillin

at a saturating concentration of 5 μg/ml and the radiolabeled PBPs were separated by SDS-PAGE and detected by fluorography. The PBP corresponding to each band is indicated on the right. Table 1 Relative amounts of PBPs in recombinant L.monocytogenes strains Protein Amount of PBP protein a   L. monocytogenes pAKB L. monocytogenes pAKB- lmo1438 PBP1 4.48 (± 0.45) 4.21 (± 0.81) PBP2 1 b 0.96 (± 0.08) PBP3 1.66 (± 0.15) 5.78 (± 0.47) c PBP4 1.67 (± 0.05) 3.2 (± 0.34) c PBP5 12.05 (± 0.42) 12.01 (± 1.03) a Average results of densitometric analysis of three independent fluorograms. b Values were normalized to the band intensity of PBP2

from L. monocytogenes pAKB, which was assigned the value of 1. c Indicates band with intensity significantly different (P < 0.05; acc. to Student's t-test) from the corresponding band of the control strain. Effect of PBP3 overproduction on growth and cell morphology of L. monocytogenes Since mutation of the lmo1438 gene did not cause HCS assay any changes in the growth and cell morphology of L. monocytogenes, the physiological role of PBP3 is unclear. To better understand the cellular function of PBP3, the effect of increased production of this protein on the growth and morphology of L. monocytogenes was examined. The growth rate of the strain overproducing

PBP3 was visibly retarded during the exponential phase of growth, when the doubling time of L. monocytogenes pAKB-lmo1438 was 116 min compared to 62 min for L. monocytogenes pAKB. However, in the stationary phase of growth the culture of L. monocytogenes pAKB-lmo1438 reached a higher OD600 value compared to the control these strain, which correlated with a significantly higher number of viable bacteria in this phase of growth (Figure 3A). Figure 3 Effect of overproduction of PBP3 on growth and morphology of L. monocytogenes. (A) Growth of L. monocytogenes pAKB (○) and L. monocytogenes pAKB-lmo1438 (•) incubated in BHI broth at 37°C following nisin induction, determined by serial dilution of the cultures and enumeration of viable cells on BHI agar. Error bars represent standard deviation from the means of three independent experiments, each performed in triplicate. (B) SEM images of L. monocytogenes pAKB (Lm pAKB) and L. monocytogenes pAKB-lmo1438 (Lm pAKB-lmo1438) cells grown overnight in BHI broth at 37°C following nisin induction. The mean cell lengths (± SD), determined by measuring 100 cells of each strain, are shown in parentheses. Bar = 2 μm. Analysis of cell morphology by scanning electron microscopy revealed that L. monocytogenes pAKB and L.