DOI: 10.5061/dryad.t1g1jwt0j
title: Salmonella enterica serovar Enteritidis EN1660 proteome spectral data

description: H-NS is a nucleoid structuring protein and global repressor of virulence
 and horizontally-acquired genes in bacteria. H-NS can interact with itself
 or with homologous proteins, but protein family diversity and regulatory
 network overlap remain poorly defined. Here we present a comprehensive
 phylogenetic analysis that revealed deep-branching clades, dispelling the
 presumption that H-NS is the progenitor of varied molecular backups. With
 few exceptions, clades are either entirely chromosomal or entirely
 plasmid-encoded proteins. On chromosomes, StpA and newly discovered HlpP
 are core genes in specific genera, whereas Hfp and newly discovered HlpC
 are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp)
 exhibit ancient and dedicated associations with plasmids, including three
 clades with fidelity for plasmid incompatibility groups H, F, or X. A
 proliferation of H-NS homologs in Erwiniaceae includes the first
 observation of potentially co-dependent H-NS forms. Conversely, the
 observed diversification of oligomerization domains may facilitate stable
 co-existence of divergent homologs in a genome. Transcriptomic and
 proteomic analysis of regulatory crosstalk in Salmonella revealed
 networked and hierarchical control of H-NS homologs. We also discovered
 that H-NS is both a repressor and activator of Salmonella Pathogenicity
 Island 1 gene expression, and both modes are restored by Sfh (HppH) in the
 absence of H-NS.
description: Cells from exponentially growing wildtype and mutant
 <i>S</i>. Enteritidis EN1660 cultures (100 ml) were harvested
 at OD<sub>600nm</sub> 0.12 – 0.15, then lysed with bead
 beating. Proteins were extracted using acetone precipitation overnight at
 -20 °C. After resolubilization in 50 mM ammonium bicarbonate buffer, 50 µg
 of protein sample was digested with trypsin (Sigma-Aldrich). After drying,
 the resulting peptides were spiked with 40 fmol/µL rabbit phosphorylase B
 in running buffer (0.1% formic acid, 3% acetonitrile) before analysis with
 a Synapt G2 HDMS (Waters) coupled to a Nanoacquity (Waters) nano-LC with
 an Acquity UPLC T3HSS column (75 mm x 200 mm). The separation was
 conducted with a gradient from 3% acetonitrile/0.1% formic acid to 45%
 acetonitrile/0.1% formic acid at a flow rate of 0.3 µL/min. A total of 1
 µg digest was injected in each run and eluting peptides were analysed in
 positive data-independent-acquisition mode (MSE) with 1s scan time. In
 low-energy MS mode data were collected at a collision energy of 4eV.
 High-energy collision energy was ramped between 18 and 42 V. Leucine
 enkephaline was measured as lock mass every 30s to maintain mass accuracy
 throughout the run.  The resulting spectra were analysed with the
 ProteinLynx Global Server (PLGS) v. 3.02 and searched against the UniProt
 <i>Salmonella enterica</i> reference proteome (UP000001014,
 accessed January 2016). Protein abundance was measured
 together with the identification using phosphorylase B as calibrant. The
 false discovery rate of protein identifications was set to 4% per run.
 i.e. proteins identified in all three biological replicates had an
 expected false discovery rate of &lt;0.001%.
description: File names correspond to <em>Salmonella
 </em>Enteritidis mutants as outlined in the manuscript. Data are in
 Waters native file format and have been compressed with 7zip (https://www.7-zip.org/). Since the data was acquired in data-independent mode (MSE) processing using ProteinLynx Global Server is recommended. Downstream analyses are open to different workflows including Ion-accounting (Waters) Mascot, Skyline etc.