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Physiology and Growth |
Division of Molecular and Genetic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, UK1
Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Sheffield S10 2TN, UK2
Author for correspondence: Jonathan G. Shaw. Tel: +44 114 271 3517. Fax: +44 114 273 9926. e-mail: J.G.Shaw{at}sheffield.ac.uk
The pathogenic neisseriae are fastidious bacteria that are only able to grow on a restricted range of carbon sources. The genome sequence of Neisseria meningitidis strain MC58 predicts the presence of a complete citric acid cycle (CAC), but there have been no detailed biochemical studies of carbon metabolism in this important pathogen. In this study, both NMR and conventional enzyme assays were used to investigate the central metabolic pathways of a serogroup B strain (K454). 13C-NMR labelling patterns of amino acids from hydrolysed cell proteins after growth with either 2- or 3-[13C]pyruvate were consistent with the operation of a complete oxidative CAC. Enzyme assays showed that cell-free extracts contained all the CAC enzymes predicted from the genome sequence, including a membrane-bound malate:quinone oxidoreductase which is present in place of the conventional NAD-linked cytoplasmic malate dehydrogenase. 1H-NMR studies showed that growth on glucose, lactate and, especially, pyruvate, resulted in the excretion of significant amounts of acetate into the culture supernatant. This occurred via the phosphotransacetylase (PTA)–acetate kinase (ACK) pathway. Extremely high specific activities of PTA (7–14 µmol min-1 mg-1) were detected in cell-free extracts, although ACK activities were much lower (46–298 nmol min-1 mg-1). Expression of PTA and ACK activities was not co-ordinately regulated during growth on combinations of carbon sources. This may be related to the presence of two ackA paralogues in N. meningitidis which are, unusually, unlinked to the pta gene.
Keywords: nuclear magnetic resonance, phosphotransacetylase–acetate kinase pathway, proton NMR, carbon-13 NMR
Abbreviations: ACK, acetate kinase; AP5A, P1,P5-di(adenosine-5’) pentaphosphate; CAC, citric acid cycle; CFE, cell-free extract; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PTA, phosphotransacetylase; TSP, trimethylsilyl propionate
a Present address: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
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