Dynamics of the Escherichia coli proteome in response to nitrogen starvation and entry into the stationary phase.

Nitrogen is needed for the biosynthesis of biomolecules including proteins and nucleic acids. In the absence of fixed nitrogen prokaryotes such as E. coli immediately ceases growth. Ammonium is the preferred nitrogen source for E. coli supporting the fastest growth rates. Under conditions of ammonium limitation, E. coli can use alternative nitrogen sources to supply ammonium ions and this reprogramming is led by the induction of the NtrC regulon. Here we used label free proteomics to determine the dynamics of E. coli proteins expression in response to ammonium starvation in both the short (30min) and the longer (60min) starvation. Protein abundances and post-translational modifications confirmed that activation of the NtrC regulon acts as the first line of defense against nitrogen starvation. The ribosome inactivating protein Rmf was induced shortly after ammonium exhaustion and this was preceded by induction of other ribosome inactivating proteins such as Hpf and RaiA supporting the hypothesis that ribosome shut-down is a key process during nitrogen limitation stress. The proteomic data revealed that growth arrest due to nitrogen starvation correlates with the accumulation of proteins involved in DNA condensation, RNA and protein catabolism and ribosome hibernation. Collectively, these proteome adaptations will result in metabolic inactive cells which are likely to exhibit multidrug tolerance.

Conserved histidine residues at the ferroxidase centre of the Campylobacter jejuni Dps protein are not strictly required for metal binding and oxidation.

Iron is an essential micronutrient for living organisms as it is involved in a broad variety of important biological processes. However, free iron inside the cell could be potentially toxic, generating hydroxyl radicals through the Fenton reaction. Dps (DNA-binding protein from starved cells) belongs to a subfamily of ferritins and can store iron atoms inside the dodecamer. The presence of a ferroxidase centre, composed of highly conserved residues, is a signature of this protein family. In this study, we analysed the role of two conserved histidine residues (H25 and H37) located at the ferroxidase centre of the Campylobacter jejuni Dps protein by replacing them with glycine residues. The C. jejuni H25G/H37G substituted variant showed reduced iron binding and ferroxidase activities in comparison with wt Dps, while DNA-binding activity remained unaffected. We also found that both CjDps wt and CjDps H25G/H37G were able to bind manganese atoms. These results indicate that the H25 and H37 residues at the ferroxidase centre of C. jejuni Dps are not strictly required for metal binding and oxidation.

Purification of the Campylobacter jejuni Dps protein assisted by its high melting temperature.

Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.