N the proton motive force and could drive a decrease in ATP synthase in order to maximise proton retention within the cytoplasm. A decrease in ATP synthase activity in cells grown at alkaline pH has been demonstrated previously [24]. Consequently, we hypothesised that the mechanism driving the observed decrease in ATP synthase activity in alkaline adapted cells is a diminished proton motive force caused by the high cell surface pH (low proton concentration) resulting from the alkaline culture media. This concept is described in a microbial attachment model introduced 11967625 by Hong and Brown [25]. In their model, they suggest that a charge-regulation effect may be induced by the environmental pH (attachment surface in their study) in proximity to the cell surface, resulting in a netAlkaline Induced Anaerobiosis in L. monocytogenesFigure 3. Evidence of stringent response (SR) induction in L. monocytogenes following alkaline adaptation. Characteristic expression of proteins previously associated with the bacterial SR was observed. A) Increased expression of the RelA synthase (a key enzyme involved in the bacterial SR), stress-related proteins (e.g. GroES, trigger factor and others), transcriptional repressor CodY, acetolactate synthase (AlsS), phosphocarrier protein (HPr), catabolite control protein A (CcpA) and decreased expression of elongation factors (EF’s) and the GTPase ObgE. Increased expression of the RelA synthase initiates production of the SR alarmone guanosine tetraphosphate (ppGpp) [39]. Chaperones stabilise proteins essential for survival during the SR and their expression can reportedly be induced by ObgE protein which is inversely correlated with ppGpp levels and cellular growth rate (as are the EF’s) [40,41,42,43]. Increased expression of AlsS, associated with branched chain amino-acid biosynthesis (BCAAS), reportedly has a role in both pH homeostasis and the bacterial SR representing a shunt of fermentation from end-product generation. Further, BCAAS is regulated by CcpA which, in turn, has been shown to complex with HPr to modulate many genes characteristic of the SR [44,45]. Similarly, CodY has been 1655472 shown to be induced as a part of the SR, is associated with regulation of .200 genes and transcriptional repression of CodY is increased in the presence of BCAA’s [42,46,47]. B) Ribosomal proteins. A global decrease in expression of ribosomal proteins, concordant with a decrease in protein synthesis, is characteristic of the bacterial SR [40,42,48]. Further, this decrease in ribosomal proteins correlates with a diminished growth rate and expression of ObgE protein [49]. C) Aminoacyl-tRNA synthetase expression is decreased, correlating with the observed decrease in ribosomal proteins and growth rate at alkaline pH [40,42]. doi:10.1371/journal.pone.0054157.gmigration of protons out of the cell. This migration of protons can shift the equilibrium of the ATP synthase phosphorylation reaction [24,25,26]. The associated MedChemExpress 4EGI-1 deficit in energy (ATP) production maybe offset by the observed increase in substrate level phosphorylation in the present study (Figure 5).Alkaline Induced Anaerobiosis in L. monocytogenesFigure 4. Proteins identified in the current study which are associated with energy metabolism. Fold change (growth at pH9.0 relative to pH7.3) lmo numbers and KEGG (http://www.genome.jp/kegg/) enzyme classification numbers are shown. doi:10.1371/journal.pone.0054157.gThe combination of these mechanisms of acidification, order POR 8 including the.N the proton motive force and could drive a decrease in ATP synthase in order to maximise proton retention within the cytoplasm. A decrease in ATP synthase activity in cells grown at alkaline pH has been demonstrated previously [24]. Consequently, we hypothesised that the mechanism driving the observed decrease in ATP synthase activity in alkaline adapted cells is a diminished proton motive force caused by the high cell surface pH (low proton concentration) resulting from the alkaline culture media. This concept is described in a microbial attachment model introduced 11967625 by Hong and Brown [25]. In their model, they suggest that a charge-regulation effect may be induced by the environmental pH (attachment surface in their study) in proximity to the cell surface, resulting in a netAlkaline Induced Anaerobiosis in L. monocytogenesFigure 3. Evidence of stringent response (SR) induction in L. monocytogenes following alkaline adaptation. Characteristic expression of proteins previously associated with the bacterial SR was observed. A) Increased expression of the RelA synthase (a key enzyme involved in the bacterial SR), stress-related proteins (e.g. GroES, trigger factor and others), transcriptional repressor CodY, acetolactate synthase (AlsS), phosphocarrier protein (HPr), catabolite control protein A (CcpA) and decreased expression of elongation factors (EF’s) and the GTPase ObgE. Increased expression of the RelA synthase initiates production of the SR alarmone guanosine tetraphosphate (ppGpp) [39]. Chaperones stabilise proteins essential for survival during the SR and their expression can reportedly be induced by ObgE protein which is inversely correlated with ppGpp levels and cellular growth rate (as are the EF’s) [40,41,42,43]. Increased expression of AlsS, associated with branched chain amino-acid biosynthesis (BCAAS), reportedly has a role in both pH homeostasis and the bacterial SR representing a shunt of fermentation from end-product generation. Further, BCAAS is regulated by CcpA which, in turn, has been shown to complex with HPr to modulate many genes characteristic of the SR [44,45]. Similarly, CodY has been 1655472 shown to be induced as a part of the SR, is associated with regulation of .200 genes and transcriptional repression of CodY is increased in the presence of BCAA’s [42,46,47]. B) Ribosomal proteins. A global decrease in expression of ribosomal proteins, concordant with a decrease in protein synthesis, is characteristic of the bacterial SR [40,42,48]. Further, this decrease in ribosomal proteins correlates with a diminished growth rate and expression of ObgE protein [49]. C) Aminoacyl-tRNA synthetase expression is decreased, correlating with the observed decrease in ribosomal proteins and growth rate at alkaline pH [40,42]. doi:10.1371/journal.pone.0054157.gmigration of protons out of the cell. This migration of protons can shift the equilibrium of the ATP synthase phosphorylation reaction [24,25,26]. The associated deficit in energy (ATP) production maybe offset by the observed increase in substrate level phosphorylation in the present study (Figure 5).Alkaline Induced Anaerobiosis in L. monocytogenesFigure 4. Proteins identified in the current study which are associated with energy metabolism. Fold change (growth at pH9.0 relative to pH7.3) lmo numbers and KEGG (http://www.genome.jp/kegg/) enzyme classification numbers are shown. doi:10.1371/journal.pone.0054157.gThe combination of these mechanisms of acidification, including the.
