Profile migration of PGA (Poly-g-glutamate) on 0.8% agarose gel electrophoresis.

<p>(a) commercial γ-PGA (Sigma Aldrich, G1049); (b) commercial PGA 3–15 kDa (Sigma Aldrich, P4636); (c) commercial PGA 15–50 kDa (Sigma Aldrich, P4886) and PGA extracted from the supernatant of <i>Bacillus subtilis</i> growth (d) after no exposure and (e) after exposure to silver nanoparticles (n-Ag), (f) to n-Ag2S or (g) to Ag lactate.</p

The poly-gamma-glutamate of <i>Bacillus subtilis</i> interacts specifically with silver nanoparticles

<div><p>For many years, silver nanoparticles, as with other antibacterial nanoparticles, have been extensively used in manufactured products. However, their fate in the environment is unclear and raises questions. We studied the fate of silver nanoparticles in the presence of bacteria under growth conditions that are similar to those found naturally in the environment (that is, bacteria in a stationary phase with low nutrient concentrations). We demonstrated that the viability and the metabolism of a gram-positive bacteria, <i>Bacillus subtilis</i>, exposed during the stationary phase is unaffected by 1 mg/L of silver nanoparticles. These results can be partly explained by a physical interaction of the poly-gamma-glutamate (PGA) secreted by <i>Bacillus subtilis</i> with the silver nanoparticles. The coating of the silver nanoparticles by the secreted PGA likely results in a loss of the bioavailability of nanoparticles and, consequently, a decrease of their biocidal effect.</p></div

In the cell-free supernatant, the soluble PGA is inversely related to the silver nanoparticle (n-Ag) concentration.

<p>(a) Representative dot blot analysis of the soluble PGA in the cell-free supernatant after incubation with n-Ag at different concentrations, ranging from 0 to 100 mg/ml. (b) Quantification of dot blot intensity normalised by the control and the amount of proteins. Error bars ± 1 standard deviation (<i>n</i> ≥ 3). Asterisks (*) indicate significant differences (<i>p</i> < 0.05).</p

Real-time quantitative polymerase chain reaction (RT-qPCR) of <i>ggt</i> and <i>pgdS</i> mRNA.

<p>The expression of <i>ggt</i> (black) and <i>pgdS</i> (white) was measured by qRT-PCR on <i>Bacillus subtilis</i> exposed to 1 mg/L of silver nanoparticles (n-Ag), 1 mg/L of n-Ag₂S or 1.8 mg/L of Ag lactate. The RNA expression is described in the Materials and Methods section. Error bars ± 1 standard deviation (<i>n</i> ≥ 3).</p

Ag K-edge EXAFS spectra for PGA mixed with Ag lactate and with Ag-NPs, and for various Ag reference compounds.

<p>The amplitude of the top two spectra was divided by 2 for better visualization of the other ones. The spectrum for PGA +AgNPs was fitted by 100% AgNPs (dotted line, <i>NSS</i> = 0.02), and PGA + Ag lactate was fitted with 38% Ag lactate (solution) + 40%Ag pectin (dotted line, <i>NSS</i> = 0.24).</p

Less PGA is extracted from growth supernatant after exposure of <i>Bacillus subtilis</i> to silver nanoparticles (n-Ag).

<p>(a) Representative dot blot of the extracted PGA. (b) Quantification of dot blot intensity after stress to <i>Bacillus subtilis</i>. The intensities were normalised by the control and the amount of proteins. Error bars ± 1 standard deviation (<i>n</i> ≥ 3). Asterisks (*) indicate significant differences vs the control (<i>p</i> < 0.05).</p

PGA interacts specifically with silver nanoparticles (n-Ag).

<p>(a) Representative dot blot of PGA after incubation of cell-free supernatant with n-Ag, n-Ag₂S or Ag lactate. (b) Quantification of dot blot. The intensity was normalised by the control and the amount of proteins. Error bars ± 1 standard deviation (<i>n</i> ≥ 3). Asterisks (*) indicate significant differences vs the control (<i>p</i> < 0.05).</p

MOESM4 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and fragment ion type, charge state, elution time as well as raw data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form

MOESM9 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio (> 5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces

MOESM5 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 5. Composition of the mixture of standard peptides