Ter adaptation to growth at alkaline pH (order BI-78D3 Figure 3; [11]). A part of 78919-13-8 thisresponse involved a shift in energy metabolism from oxidative to substrate level phosphorylation (Figures 4 and 5). Altering the flow of substrates through these pathways could serve a number of functions under alkaline conditions. The pentose phosphate shunt increases production of reducing equivalents (NADH) directed at fatty acid biosynthesis, the electron transport chain (ETC) and a wide range of other cellular processes. Increased proteins associated with fatty acid biosynthesis and degradation was observed in the present study; however, it was coupled to a decrease in other proteins also associated with biosynthesis of fatty acids (Table S1). While fatty acids are known to have a role in the pH tolerance response of L. monocytogenes, it is reported that the type, rather 
than number, of fatty acid is what imparts the protective effect [21]. On this basis the observed differences in protein abundances associated with fatty acid biosynthesis may reflect the type of fatty acids being produced and/or degraded, however this was not further investigated experimentally. Importation of sugars via the phosphotransferase system (PTS) is shown to be important for buffering of the cell cytoplasm [22], while increasing substrates for glycolysis. Glycolysis, the pentose phosphate shunt and PTS system produce by-products that are associated with the electron transport chain. This multi-step energy generating system involves a number of protein components that transfer electrons from the initial NADH and succinate donors (generated by the pentose phosphate/glycolysis pathways, and the limited fatty acid degradation observed in the current study), culminating in energy production by an ATP synthase powered by a proton motive force [23]. However, in the present study, decreases in ubiquinone biosynthetic enzymes wereAlkaline Induced Anaerobiosis in L. monocytogenesFigure 2. Protein groups identified in the current study previously associated with alkaline pH homeostasis [6,11,21,26,34,35,36,37,38]. Broad (A) and specific (B) functional grouping categories based on the JCVI-CMR L. monocytogenes EGD-e functional ontology system (http://cmr.jcvi.org/cgibin/CMR/shared/RoleList.cgi). doi:10.1371/journal.pone.0054157.gdetected, along with decreased abundance of proteins associated with ATP-proton motive force (e.g. F0F1 ATPase subunits lmo0092, 0088, 2530, 2532 and 2528) and electron transport chain 15755315 complexes one, two and five (e.g. NADH dehydrogenase lmo2638 and 2389, and fumarate reductase lmo0355; Figure 4 and 5). A decrease in proton motive force was supported by the increased expression of PTS system proteins (e.g. lmo0507, 1719, 2335, 1002 and 1003; Figure 4). Maintenance of intracellular pH in L. monocytogenes was shown by Shabala et al [22] to be coupled to two glucose transport systems: a low-affinity proton motive forcedriven system and a high affinity PTS system. As such, should proton motive force be forcibly diminished it could be expected that proteins associated with the PTS-mediated glucose transport system would increase to compensate, as shown in Figure 4. A diminished ATP-proton motive force would appear to oppose, to some extent, any cytoplasmic acidification process, as the proton pump (driven by the proton motive force) expels protons in the generation of energy (ATP) via an ATP synthase.Considering the decreased proton motive force and associated protein abundan.Ter adaptation to growth at alkaline pH (Figure 3; [11]). A part of thisresponse involved a shift in energy metabolism from oxidative to substrate level phosphorylation (Figures 4 and 5). Altering the flow of substrates through these pathways could serve a number of functions under alkaline conditions. The pentose phosphate shunt increases production of reducing equivalents (NADH) directed at fatty acid biosynthesis, the electron transport chain (ETC) and a wide range of other cellular processes. Increased proteins associated with fatty acid biosynthesis and degradation was observed in the present study; however, it was coupled to a decrease in other proteins also associated with biosynthesis of fatty acids (Table S1). While fatty acids are known to have a role in the pH tolerance response of L. monocytogenes, it is reported that the type, rather than number, of fatty acid is what imparts the protective effect [21]. On this basis the observed differences in protein abundances associated with fatty acid biosynthesis may reflect the type of fatty acids being produced and/or degraded, however this was not further investigated experimentally. Importation of sugars via the phosphotransferase system (PTS) is shown to be important for buffering of the cell cytoplasm [22], while increasing substrates for glycolysis. Glycolysis, the pentose phosphate shunt and PTS system produce by-products that are associated with the electron transport chain. This multi-step energy generating system involves a number of protein components that transfer electrons from the initial NADH and succinate donors (generated by the pentose phosphate/glycolysis pathways, and the limited fatty acid degradation observed in the current study), culminating in energy production by an ATP synthase powered by a proton motive force [23]. However, in the present study, decreases in ubiquinone biosynthetic enzymes wereAlkaline Induced Anaerobiosis in L. monocytogenesFigure 2. Protein groups identified in the current study previously associated with alkaline pH homeostasis [6,11,21,26,34,35,36,37,38]. Broad (A) and specific (B) functional grouping categories based on the JCVI-CMR L. monocytogenes EGD-e functional ontology system (http://cmr.jcvi.org/cgibin/CMR/shared/RoleList.cgi). doi:10.1371/journal.pone.0054157.gdetected, along with decreased abundance of proteins associated with ATP-proton motive force (e.g. F0F1 ATPase subunits lmo0092, 0088, 2530, 2532 and 2528) and electron transport chain 15755315 complexes one, two and five (e.g. NADH dehydrogenase lmo2638 and 2389, and fumarate reductase lmo0355; Figure 4 and 5). A decrease in proton motive force was supported by the increased expression of PTS system proteins (e.g. lmo0507, 1719, 2335, 1002 and 1003; Figure 4). Maintenance of intracellular pH in L. monocytogenes was shown by Shabala et al [22] to be coupled to two glucose transport systems: a low-affinity proton motive forcedriven system and a high affinity PTS system. As such, should proton motive force be forcibly diminished it could be expected that proteins associated with the PTS-mediated glucose transport system would increase to compensate, as shown in Figure 4. A diminished ATP-proton motive force would 
appear to oppose, to some extent, any cytoplasmic acidification process, as the proton pump (driven by the proton motive force) expels protons in the generation of energy (ATP) via an ATP synthase.Considering the decreased proton motive force and associated protein abundan.