Agarose gel electrophoresis analysis of HindIII-digested fosmid clones before and after conjugation from <i>E. coli</i> S17-1 to <i>P. fluorescens::</i>Tn<i>RS48</i> or <i>X. campestris::</i>Tn<i>RS48</i>.

<p>Lane 1, plasmid 62 before transfer, and lanes 2 and 3 after transformation to <i>E. coli</i> from <i>P. fluorescens</i> and <i>X. campestris</i>, respectively. Lane 4, plasmid 83 before conjugal transfer, and after transformation to <i>E. coli</i> from <i>P. fluorescens</i> (lanes 5-7) and <i>X. campestris</i> (lanes 8 and 9). Lane 10, plasmid 37 before transfer, and after transformation to <i>E. coli</i> from <i>P. fluorescens</i>. (lanes 11 and 12) and <i>X. campestris</i> (lane 13). S: Molecular weight standard (Fermentas).</p

Agarose gel electrophoresis analysis of HindIII-digested fosmid clones that have been conjugatively transferred from <i>E. coli</i> DH10B/pTA-Mob to <i>P. fluorescens::</i>Tn<i>RS48</i>.

<p>Lane 1: plasmid 37 before transfer and lanes 2-4 after conjugation. Lane 5: Plasmid 83 before transfer and lanes 6–8 after conjugation. S: Molecular weight standard (NEB).</p

Map of the mobilization helper plasmid pTA-Mob with relevant regions depicted.

<p>Gm^r, gentamycin resistance gene; <i>rep</i>, pBBR1 replication protein gene; <i>ori</i>; pBBR1 replication origin; (<i>trfA</i>), replication initiation protein gene from the RK2 replicon, this replicon is not active due to lack of RK2 replication origin <i>oriV</i>; Tra1 and Tra2, regions containing the <i>tra</i> genes necessary for conjugative transfer of <i>oriT</i> containing plasmids; <i>parABCDE</i>, stabilization region encoding the gene products ParA, B, C, D and E; Ctl, central control operon of RK2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090372#pone.0090372-Pansegrau1" target="_blank">[6]</a>.</p

Decay kinetics for <i>gm-csf</i> and <i>ompA-gm-csf</i> transcripts expressed from <i>P_tac</i>.

<p><b>A.</b><i>gm-csf</i> and <i>ompA</i>-<i>gm-csf</i> genes was achieved by the inducer wash-out. <b>B.</b> Rifampicin was used for transcription inhibition. For both A and B: the larger plot shows <i>gm-csf</i> and <i>ompA</i>-<i>gm-csf</i> transcript amounts at time point zero arbitrary set to one. In the upper right corner the decay curves are represented with all values relative to <i>gm-csf</i> transcript level at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066429#pone.0066429.e008" target="_blank">Eqn 4</a> (Material and Methods). Error bars show the deviation between at least three technical recurrences. RQ: relative quantification, AU: arbitrary units.</p

Kinetics of <i>gm-csf</i> and <i>ompA-gm-csf</i> transcript decay, expressed from <b><i>Pm</i></b>.

<p>The larger plot shows decay kinetics with both <i>gm-csf</i> and <i>ompA-gm-csf</i> transcript levels at time point zero arbitrarily set to one. The smaller plot shown in the upper right corner represents all transcript data relative to the value of <i>gm-csf</i> at time point zero (arbitrarily set to one). Solid lines represent the best fit to the data calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066429#pone.0066429.e008" target="_blank">Eqn 4</a> (Material and Methods). Error bars show the deviation between two biological recurrences. RQ: relative quantification, AU: arbitrary units.</p

PCR and qRT PCR primers.

1<p>Km^r: kanamycin resistance gene.</p

The <i>bla</i> gene expression in DH5α (pBSP1bla) and DH5α (pBSP1bla-C19).

<p><b>A.</b> Determination of accumulated transcript levels and enzymatic activity of β-lactamase, with all values relative to the pBSP1bla level (arbitrarily set to one). <b>B.</b> Total β-lactamase production of DH5α (pBSP1bla) (denoted as wt) and DH5α (pBSP1bla-C19) (denoted as C19) was visualized by Western blotting and total cell samples were loaded in dilution series of decreasing total protein amount (µg). The size of the mature β-lactamase protein is approximately 29 kDa.</p

Accumulation and decay of <i>IL1RN_S</i> transcripts.

<p>The respective genes were expressed from either the T7 or the <i>Pm</i> promoter. <b>A.</b> For both T7 and <i>Pm</i> promoter generated transcripts the amounts are given relative to the value at time point 60 minutes (arbitrarily set to one). The amounts of <i>IL1RN_S</i> transcript generated through the T7 system was increased about 10-fold compared to those of the <i>Pm</i> system (after 60 minutes). Solid lines represent the best fit to the data, calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066429#pone.0066429.e005" target="_blank">Eqn 2</a> (Material and Methods). In case of the T7 system, only time points 15, 20, 40 and 60 minutes were included for the generation of the transcript accumulation curve. <b>B.</b> Parallel determination of decay for <i>Pm</i> and T7 generated transcripts. For both systems all transcript amounts are presented relative to <i>IL1RN_S</i> transcript level at time zero (arbitrarily set to one). Error bars show the deviation between three biological recurrences. Solid lines represent the best fit to the data calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066429#pone.0066429.e008" target="_blank">Eqn 4</a> (Material and Methods). RQ: relative quantification, AU: arbitrary units.</p

Decay kinetics of two <i>bla</i> transcripts with different 5′ UTR sequences.

<p>The respective genes were expressed from <i>Pm</i> and contained the DNA sequence of either the wild type 5′-UTR or the LV-2 variant. The larger plot describes decay kinetics where the level of the two <i>bla</i> transcripts is arbitrary set to one at time point zero. In the upper right corner the transcript amounts are represented with all values relative to the wild type 5′-UTR; arbitrary set to one at time point zero. Solid lines represent the best fit to the data calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066429#pone.0066429.e008" target="_blank">Eqn 4</a> (Material and Methods). Error bars show the deviation between three biological recurrences. RQ: relative quantification, AU: arbitrary units.</p

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1β release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage