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Abstract

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine
respiratory tract lacking enzymes of the oxidative branch of the
tricarboxylic acid (TCA) cycle. We previously claimed that A.
pleuropneumoniae instead uses the reductive branch in order to generate
energy and metabolites. Here, we show that bicarbonate and oxaloacetate
supported anaerobic growth of A. pleuropneumoniae. Isotope mass
spectrometry revealed heterotrophic fixation of carbon from stable
isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed
by nano-scale secondary ion mass spectrometry at a single-cell level. By
gas chromatography-combustion-isotope ratio mass spectrometry we could
further show that the labeled carbon atom is mainly incorporated into
the amino acids aspartate and lysine, which are derived from the TCA
metabolite oxaloacetate. We therefore suggest that carbon fixation
occurs at the interface of glycolysis and the reductive branch of the
TCA cycle. The heme precursor delta-aminolevulinic acid supported growth
of A. pleuropneumoniae, similar to bicarbonate, implying that
anaplerotic carbon fixation is needed for heme synthesis. However,
deletion of potential carbon-fixing enzymes, including PEP-carboxylase
(PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate
decarboxylase, as well as various combinations thereof, did not affect
carbon fixation. Interestingly, generation of a deletion mutant lacking
all four enzymes was not possible, suggesting that carbon fixation in A.
pleuropneumoniae is an essential metabolic pathway controlled by a
redundant set of enzymes. A double deletion mutant lacking PEPC and
PEPCK was not impaired in carbon fixation in vitro but showed reduction
of virulence in a pig infection model.