Resuscitation of VBNC <i>Escherichia coli</i> O157:H7 cells induced by HPCD treatment.

<p>(A) 5 MPa and 25°C for 40 min; (B) 5 MPa and 31°C for 30 min; (C) 5 MPa and 34°C for 28 min. Resuscitation samples were cultured in TSB and incubated at 37°C for 6 h and 24 h. Shown are plate counts for each 10-fold serial dilution (from 10⁰ to 10⁻⁴) sample.</p

Entry of <i>Escherichia coli</i> O157:H7 into a VBNC state by HPCD treatment.

<p>(A) 5 MPa, 25°C; (B) 5 MPa, 31°C; (C) 5 MPa, 34°C; (D) 5 MPa, 37°C. Shown are plate counts (▴) on tryptic soy agar (TSA), and viable cell counts (•) and total cell counts (▪) by the Live/Dead staining method.</p

Transmission electron micrograph of <i>Escherichia coli</i> O157:H7.

<p>(A) the exponential-phase cells; (B) VBNC cells induced by HPCD treatment at 5 MPa and 25°C for 40 min; (C) resuscitated cells cultured in TSB for 6 h; (D) resuscitated cells cultured in TSB for 24 h. Left images, magnification of × 25,000; right images, except in panel A, magnification of × 80,000; right image of panel A, magnification of × 60,000.</p

Scanning electron micrograph (magnification of × 10,000) of <i>Escherichia coli</i> O157:H7.

<p>(A) the exponential-phase cells; (B) VBNC cells induced by HPCD treatment at 5 MPa and 25°C for 40 min; (C) resuscitated cells cultured in TSB for 6 h; (D) resuscitated cells cultured in TSB for 24 h.</p

Enzymatic activities of <i>Escherichia coli</i> O157:H7 measured by the API ZYM system.

<p>(A) the exponential-phase cells; (B) VBNC cells treated by HPCD at 5 MPa and 25°C for 40 min; (C) VBNC cells treated by HPCD at 5 MPa and 31°C for 30 min; (D) VBNC cells treated by HPCD at 5 MPa and 34°C for 28 min; (E) VBNC cells treated by HPCD at 5 MPa and 37°C for 25 min.</p

Resistance of VBNC <i>Escherichia coli</i> O157:H7 cells to sonication.

<p>The VBNC <i>E. coli</i> O157:H7 cells were induced by HPCD treatment at 5 MPa and 25°C for 40 min.</p

Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex

<div><p>Upon exposure to exogenous pediocin-like bacteriocins, immunity proteins specifically bind to the target receptor of the mannose phosphotransferase system components (man-PTS IIC and IID), therefore preventing bacterial cell death. However, the specific recognition of immunity proteins and its associated target receptors remains poorly understood. In this study, we constructed hybrid receptors to identify the domains of IIC and/or IID recognized by the immunity protein PedB, which confers immunity to pediocin PA-1. Using <i>Lactobacillus plantarum</i> man-PTS EII mutant W903, the IICD components of four pediocin PA-1-sensitive strains (<i>L</i>. <i>plantarum</i> WQ0815, <i>Leuconostoc mesenteroides</i> 05–43, <i>Lactobacillus salivarius</i> REN and <i>Lactobacillus acidophilus</i> 05–172) were respectively co-expressed with the immunity protein PedB. Well-diffusions assays showed that only the complex formed by LpIICD from <i>L</i>. <i>plantarum</i> WQ0815 with pediocin PA-1 could be recognized by PedB. In addition, a two-step PCR approach was used to construct hybrid receptors by combining LpIIC or LpIID recognized by PedB with the other three heterologous IID or IIC compounds unrecognized by PedB, respectively. The results showed that all six hybrid receptors were recognized by pediocin PA-1. However, when IIC or IID of <i>L</i>. <i>plantarum</i> WQ0815 was replaced with any corresponding IIC or IID component from <i>L</i>. <i>mesenteroides</i> 05–43, <i>L</i>. <i>salivarius</i> REN and <i>L</i>. <i>acidophilus</i> 05–172, all the hybrid receptors could not be recognized by PedB. Taken altogether, we concluded that both IIC and IID components of the mannose phosphotransferase system play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB.</p></div

Sensitivity of <i>L</i>. <i>plantarum</i> W903 derivatives harboring gene <i>IICD</i> or <i>IICD</i> and <i>pedB</i> to pediocin PA-1.

<p>Sensitivity of <i>L</i>. <i>plantarum</i> W903 derivatives harboring gene <i>IICD</i> or <i>IICD</i> and <i>pedB</i> to pediocin PA-1.</p

Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex - Fig 4

<p><b>Multiple sequence alignments (A) and phylogenetic clustering (B) of IIC and IID proteins from <i>L</i>. <i>plantarum</i>, <i>L</i>. <i>mesenteroides</i>, <i>L</i>. <i>salivarius</i>, <i>L</i>. <i>acidophilus</i> and <i>P</i>. <i>acidilactici</i>.</b> Transmembrane helix (TMhelix), extracellular and intracellular regions were determined by using TMHMM v. 2.0 software. An asterisk, two dots, and one dot indicated decreasing degrees of conservation. The conserved motifs GGQGxxG and GG[D/K]FxxxG in the extracellular loop region are indicated by a grey background. The residues from <i>L</i>. <i>plantarum</i> and <i>P</i>. <i>acidilactici</i> in the intracellular regions are indicated by boxes. Sequence alignments and phylogenetic trees were constructed by using MUSCLE v. 3.8.31 software with default settings (<a href="http://www.ebi.ac.uk/Tools/msa/muscle/" target="_blank">http://www.ebi.ac.uk/Tools/msa/muscle/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164973#pone.0164973.ref027" target="_blank">27</a>].</p

PCR primers for amplifying genes <i>IIC</i> and <i>IID</i>.

<p>PCR primers for amplifying genes <i>IIC</i> and <i>IID</i>.</p