Cell length and cell wall thickness of <i>L. monocytogenes</i> strains.

a<p>Mean cell lengths were determined by measuring 100 cells of each strain grown in the presence (+ PenG) or absence of 0.09 µg/ml penicillin G. The analysis was performed using two independently prepared samples. From each independent sample, equal numbers of cells were analyzed ± standard deviation (SD).</p>b<p>Mean cell wall thickness was determined by measurement of 20 cells of each strain grown in the presence (+ PenG) or absence of 0.09 µg/ml penicillin G. The analysis was performed using two independently prepared samples. From each independent sample, equal numbers of cells were analyzed ± standard deviation (SD).</p>c<p>Significant differences following growth in the presence and absence of penicillin G (Student's t-test; P<0.05).</p>d<p>Significant differences between the studied strains (Student's t-test; P<0.05).</p

Content of murein hydrolases in various cellular compartments of <i>L. monocytogenes</i> strains.

<p>Zymography analysis of proteins from the cytoplasm (A), cytoplasmic membrane (B), cell wall fraction (C) and culture supernatant (D) were performed for the wild-type EGD strain grown without (<i>Lm</i> EGD) and with (<i>Lm</i> EGD + penG) penicillin G, and the <i>Δ</i><i>fri</i> mutant strain grown without (<i>Lm</i> Δ<i>fri</i>) and with (<i>Lm</i> Δ<i>fri</i> + penG) the same antibiotic. Equivalent quantities of proteins isolated from each fraction were subjected to zymographic analysis in SDS-polyacrylamide gels. MWM – prestained Protein Molecular Weight Marker. The relative amounts of murein hydrolases estimated on the basis of the densitometric analysis are shown in the lower panel. Values in each analyzed compartment were normalized to the total intensity of murein hydrolases from the wild-type EGD strain grown without the antibiotic, which was assigned the value of 100%. The presented results are mean values from the analysis of three independent protein isolations ± the standard deviation. <i>^a</i> Significant differences following growth in the presence and absence of penicillin G (Student's t-test; P<0.05). <i>^b</i> Significant differences between the studied strains (Student's t-test; P<0.05).</p

Critical Role of a Ferritin-Like Protein in the Control of <i>Listeria monocytogenes</i> Cell Envelope Structure and Stability under β-lactam Pressure

<div><p>The human pathogen <i>Listeria monocytogenes</i> is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, <i>L. monocytogenes</i> is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of <i>L. monocytogenes</i> tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in <i>L. monocytogenes</i>, but its function in this process remains unknown. The present study was undertaken to improve our understanding of <i>L. monocytogenes</i> tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type <i>L. monocytogenes</i> and a <i>fri</i> deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of <i>L. monocytogenes</i> grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the <i>L. monocytogenes</i><i>Δ</i><i>fri</i> mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of <i>L. monocytogenes</i> cell envelope structure and stability under β-lactam pressure.</p></div

Integrity of the cell wall of <i>L. monocytogenes</i> strains.

<p>Cells of the wild-type (<i>Lm</i> EGD) and <i>Δ</i><i>fri</i> mutant (<i>Lm </i><i>Δ</i><i>fri</i>) strains grown in the absence or presence of penicillin G (+penG) were incubated with lysozyme (10 U/ml) at 37°C for 90 min. At selected intervals, the OD₆₀₀ of the cell suspensions was determined. Error bars represent the standard deviation from three independent experiments, each performed in triplicate.</p

Cell wall TA content in <i>L. monocytogenes</i> strains.

a<p>The results are the average of three independent experiments, each performed with separate cell wall preparations of the strains grown in the presence (+ PenG) or absence of 0.09 µg/ml penicillin G ± the standard deviation (SD).</p>b<p>Significant differences following growth in the presence and absence of penicillin G (Student's t-test; P<0.05).</p>c<p>Significant differences between the studied strains (Student's t-test; P<0.05).</p

Expression of promoter-<i>lacZ</i> fusions in <i>L. monocytogenes</i> strains.

a<p>The expression of promoter-<i>lacZ</i> fusions in response to the addition of 0.09 µg/ml penicillin G (+ PenG) or in the absence of the antibiotic was determined by β-galactosidase assays. Specific β-galactosidase activity was measured for wild-type (EGD) or Δ<i>fri</i> mutant cells containing promoter-<i>lacZ</i> fusions, grown in the presence or absence of the antibiotic. The results are the average of three independent experiments, each performed in triplicate ± standard deviation.</p>b<p>Genes that are organized in an operon (according to Toledo-Arana et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077808#pone.0077808-ToledoArana1" target="_blank">[66]</a>).</p>c<p>Significant differences following growth in the presence and absence of penicillin G (Student's t-test; P<0.05).</p>d<p>Significant differences between the studied strains (Student's t-test; P<0.05).</p

Cell morphology of <i>L. monocytogenes</i> strains.

<p>Scanning electron micrographs (A) and transmission electron micrographs (B) of wild-type (<i>Lm</i> EGD) and <i>Δ</i><i>fri</i> mutant (<i>Lm </i><i>Δ</i><i>fri</i>) cells grown in the absence or presence of penicillin G (+penG). Bars indicate 2 µm in the scanning electron micrographs and 200 nm in the transmission electron micrographs. The presented micrographs are representative of two independent sample preparations.</p

Oligonucleotide primers used in this study.

a<p>The sequence in boldface type is the EcoRI restriction enzyme site.</p>b<p>The sequence in boldface type is the BamHI restriction enzyme site.</p>c<p>The sequence in boldface type is the SmaI restriction enzyme site.</p