Corrigendum: Directionality of substrate translocation of the hemolysin A Type I secretion system

Scientific Reports 5: Article number: 12470; published online: 27 July 2015; updated: 08 January 2018 In this Article, figure 1 contains errors. Errors were made during the preparation of figure 1: the same HylD blot was accidentally used in figures 1 and 2. The correct Figure 1 appears below.</jats:p

Cloning and expression of selected ABC transporters from the Arabidopsis thaliana ABCG family in Pichia pastoris.

Phytohormones play a major role in plant growth and development. They are in most cases not synthesized in their target location and hence need to be transported to the site of action, by for instance ATP-binding cassette transporters. Within the ATP-binding cassette transporter family, Pleiotropic Drug Resistance transporters are known to be involved in phytohormone transport. Interestingly, PDRs are only present in plants and fungi. In contrast to fungi, there are few biochemical studies of plant PDRs and one major reason is that suitable overexpression systems have not been identified. In this study, we evaluate the expression system Pichia pastoris for heterologous overexpression of PDR genes of the model plant Arabidopsis thaliana. We successfully cloned and expressed the potential phytohormone transporters PDR2 and PDR8 in P. pastoris. Sucrose gradient centrifugation confirmed that the overexpressed proteins were correctly targeted to the plasma membrane of P. pastoris and initial functional studies demonstrated ATPase activity for WBC1. However, difficulties in cloning and heterologous overexpression might be particular obstacles of the PDR family, since cloning and overexpression of White Brown Complex 1, a half-size transporter of the same ABCG subfamily with comparable domain organization, was more easily achieved. We present strategies and highlight critical factors to successfully clone plant PDR genes and heterologously expressed in P. pastoris

SYTOx green assay to visualize pore formation mediated by nisin.

<p>The NZ9000Erm strain (black line), NZ9000NisI strain (blue line) and the NZ9000NisIΔ22 strain (red line) were grown and incubated with the SYTOX green dye. The fluorescence signal was monitored online using a fluorolog (Horiba III). After 400 seconds a stable baseline was reached and nisin was added (A) 10 nM (B) 30 nM and (C) 1000 nM. The addition of nisin is indicated with an arrow. The rapid increase of the fluorescence signal indicated pore formation. The data are representatives of at least three independent measurements.</p

Western blot analysis using a polyclonal NisI antibody (A).

<p>Shown are the <i>L. lactis</i> strains: NZ9000Erm, NZ9000NisI and NZ9000NisIΔ22 strain. <b>IC₅₀ determination of different strains (B)</b>. Growth inhibition experiments were performed with nisin using different strains. Black line: NZ9000Erm strain; blue line: NZ9000NisI strain; red line: NZ9000NisIΔ22 strain. Data was fitted and evaluated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102246#pone.0102246.e001" target="_blank">equation (1</a>). Each experiment was performed at least in triplicates.</p

Phenotype visualisation of <i>L. lactis</i> cells using the NZ9000Erm, NZ9000NisI and NZ9000NisIΔ22 strain.

<p>The different strains were grown until exponential phase (OD₆₀₀ = 0.5). During growth, different concentrations of nisin were added (0, 1, 10 and 30 nM). The cells were transferred and fixed onto a coverslide and the cells were visualised using a Nikon eclipse Ti microscope. The chains were counted and categorized in different classes. Class 1 consisted of 1–2 cocci (black bar), class 2 consisted of 3–5 cells (grey bar), class 3 consisted of 6–10 cells (dark grey bar), class 4 comprised of 11–20 cells (light grey) and class 5 comprised of >20 cells. For each sample the number of counted cells per area was >50. In total, after scanning five different areas at least >500 cell chains were observed.</p

Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

<div><p>Nisin, a 3.4 kDa antimicrobial peptide produced by some <i>Lactococcus lactis</i> strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, <i>Lactococcus lactis</i> is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC₅₀ of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the <i>Lactococcus lactis</i> cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.</p></div

Growth recovery assay.

<p>The different strains were incubated for 1 (•), 2 (▪), 3(▴), 4(▾) and 5(⧫) hours at an OD₆₀₀ of 0.1 with nisin at a concentration which represents 10-fold the IC₅₀ determined, 100 nM, 300 nM and 600 nM for the NZ9000Erm (A), NZ9000NisIΔ22 (B) and the NZ9000NisI (C) strains, respectively. The cells were separated from the growth media by centrifugation and extensively washed with media to remove the remaining nisin. Afterwards the cells were transferred into fresh medium at a final OD₆₀₀ of 0.1 and the growth was monitored by measuring the optical density at 600 nm. As a control (○) the corresponding strains without the addition of nisin during pre-incubation were used. Each experiment was performed 4 times. Within the different experiments, the interval of recovery comprised between 5 and 8 hours. Furthermore the end point OD₆₀₀ (after 15 hours growth) was in a range of 65–100% recovery ability (compared with the end point OD₆₀₀ of the control). To control the number of cells surviving the incubation with high nisin concentrations, the resuspended cells were striked out on GM17 agar plates. The number of colonies on these plates resemble the total number of living cell in the cell suspension with an OD₆₀₀ of 0.1. A normalisation of the total cell number between the strains NZ9000Erm, NZ9000NisI and NZ9000NisIΔ22 shows the relative distribution depending on the living cells (D). The NZ9000 NisI after 1 h incubation time is set as 100% (total cell number: 261.866±32.809) and reflects the 1.4% of surviving cells compared to the control (total cell number: 24.800.000±1.844.776). Longer incubation times lead to survival rates of 20% for NZ9000NisI. Even fewer cells, only 0.1%, survived for the NZ9000Erm and NZ9000NisIΔ22 strains, when compared to the control. The error bars indicating the standard deviation of three independent experiments.</p

Shaping the lipid composition of bacterial membranes for membrane protein production

Contains fulltext : 209080.pdf (publisher's version ) (Open Access

IC₅₀ values determined for the different strains.

<p>IC₅₀ values determined for the different strains.</p

Analysis of the Bile Salt Export Pump (<i>ABCB11</i>) Interactome Employing Complementary Approaches

<div><p>The bile salt export pump (BSEP, <i>ABCB11</i>) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by <i>in vitro</i> interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.</p></div