Regenerating reactions downstream of pyruvate. In our previous studies [6], exposure of

Regenerating reactions downstream of pyruvate. In our previous studies [6], exposure of S. Val of SMARTA following Lm-gp61 or LCMV infection is determined within aureus to linoleic acid caused downregulated transcription of the mtlABFD locus, which suggests, either that reduced levels of intracellular Mtl is a preferred metabolic state following exposure to AFAs, or that lower amounts of Mtl-1-P arising from metabolism (concomitantly regenerating NADH) limited induction of the operon. A potential explanation for the reduced AFA survival of the mtlD mutant is its reduced adaptive capacity due to an inability to metabolise Mtl. The near wild-type AFA survival of the mtlABFD operon mutantargues against this Mtl metabolism hypothesis, however, unless there is an alternative metabolic reserve. 3-phosphoglycerate could serve as just such an alternative metabolic source and substrate for regenerating NADH, and of note there is 3-fold reduced 3-PGA in the mtlD mutant. Metabolite Title Loaded From File analysis of the S. aureus mtlD mutant, when compared to the mtlABFD transport mutant and the parental strain, revealed that 14 of the total Mtl that accumulated intracellularly was not phosphorylated. Since the EIIMtl mannitol transporter (encoded by mtlA) phosphorylates the imported Mtl and since the Mtl-1-Pdehydrogenase activity is ablated in the mtlD mutant, the conversion of Mtl-1-P to Mtl in the mtlD mutant is likely to arise from phosphotransferase reactions as described by Saier and Newman [42]. Alternatively, an undescribed phosphatase activity might account for the presence of Mtl. In Lactobacillus plantarum a hypothetical phosphatase activity of EIIMtl was proposed to explain the apearance of Mtl in engineered strains [43]. In the study of Mtl overproducing strains of L. lactis a Mtl-1-phosphatase activity was proposed to explain the presence of unphosphorylated Mtl, where mtlA was absent, and thus an EIIMtl activity, could not be present. Analysis of the metabolites of growing cells of the S. aureus mtlD mutant cultured in BHI broth, when compared to the mtlABFD transport mutant and the parental strain, revealed further differences aside from sugar alcohol content (Table S1). These metabolite changes e.g. aminoadipic acid, 3-phosphoglycerate, hydroxypentanoic acid, heptanoic acid and tetradecanoylglycerol, do not indicate a clearly defined mechanistic explanation for decreased AFA MIC. Growth of the mtlD mutant was strongly retarded in media containing mannitol, highlighting the deleterious effects resulting from the likely unrestricted accumulation of Mtl/Mtl-1-P. A direct link between the intracellular accumulation of Mtl/Mtl-1-P and reduced resistance to AFAs in S. aureus currently lacks an evidencebased mechanism. However, several features of the mtlD phenotype could result from a membrane-associated effect. Alcohol has a well-described potentiating mechanism with respect to AFAs and their membrane activity [13], since it is capable of solubilising membrane lipids due to its polarity and lipophilicity. Intracellularly accumulated sugar alcohol, Mtl, might act similarly to potentiate AFA action, since it was demonstrated in this study that Mtl acted synergistically with linoleic acid when added externallyS. aureus Mannitol Utilisation and Survivalin BHI agar. Two further phenotypes point towards a membranespecific alteration in the mtlD mutant; the reduced growth rate that was oberved for the mtlD mutant at 25uC and the reduced MIC for H2O2 which did not result from differences in catalase specific activity (data not shown).Regenerating reactions downstream of pyruvate. In our previous studies [6], exposure of S. aureus to linoleic acid caused downregulated transcription of the mtlABFD locus, which suggests, either that reduced levels of intracellular Mtl is a preferred metabolic state following exposure to AFAs, or that lower amounts of Mtl-1-P arising from metabolism (concomitantly regenerating NADH) limited induction of the operon. A potential explanation for the reduced AFA survival of the mtlD mutant is its reduced adaptive capacity due to an inability to metabolise Mtl. The near wild-type AFA survival of the mtlABFD operon mutantargues against this Mtl metabolism hypothesis, however, unless there is an alternative metabolic reserve. 3-phosphoglycerate could serve as just such an alternative metabolic source and substrate for regenerating NADH, and of note there is 3-fold reduced 3-PGA in the mtlD mutant. Metabolite analysis of the S. aureus mtlD mutant, when compared to the mtlABFD transport mutant and the parental strain, revealed that 14 of the total Mtl that accumulated intracellularly was not phosphorylated. Since the EIIMtl mannitol transporter (encoded by mtlA) phosphorylates the imported Mtl and since the Mtl-1-Pdehydrogenase activity is ablated in the mtlD mutant, the conversion of Mtl-1-P to Mtl in the mtlD mutant is likely to arise from phosphotransferase reactions as described by Saier and Newman [42]. Alternatively, an undescribed phosphatase activity might account for the presence of Mtl. In Lactobacillus plantarum a hypothetical phosphatase activity of EIIMtl was proposed to explain the apearance of Mtl in engineered strains [43]. In the study of Mtl overproducing strains of L. lactis a Mtl-1-phosphatase activity was proposed to explain the presence of unphosphorylated Mtl, where mtlA was absent, and thus an EIIMtl activity, could not be present. Analysis of the metabolites of growing cells of the S. aureus mtlD mutant cultured in BHI broth, when compared to the mtlABFD transport mutant and the parental strain, revealed further differences aside from sugar alcohol content (Table S1). These metabolite changes e.g. aminoadipic acid, 3-phosphoglycerate, hydroxypentanoic acid, heptanoic acid and tetradecanoylglycerol, do not indicate a clearly defined mechanistic explanation for decreased AFA MIC. Growth of the mtlD mutant was strongly retarded in media containing mannitol, highlighting the deleterious effects resulting from the likely unrestricted accumulation of Mtl/Mtl-1-P. A direct link between the intracellular accumulation of Mtl/Mtl-1-P and reduced resistance to AFAs in S. aureus currently lacks an evidencebased mechanism. However, several features of the mtlD phenotype could result from a membrane-associated effect. Alcohol has a well-described potentiating mechanism with respect to AFAs and their membrane activity [13], since it is capable of solubilising membrane lipids due to its polarity and lipophilicity. Intracellularly accumulated sugar alcohol, Mtl, might act similarly to potentiate AFA action, since it was demonstrated in this study that Mtl acted synergistically with linoleic acid when added externallyS. aureus Mannitol Utilisation and Survivalin BHI agar. Two further phenotypes point towards a membranespecific alteration in the mtlD mutant; the reduced growth rate that was oberved for the mtlD mutant at 25uC and the reduced MIC for H2O2 which did not result from differences in catalase specific activity (data not shown).