Journal of Bacteriology, February 2001, p. 1038-1046, Vol. 183, No. 3
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.3.1038-1046.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
-Hydroxybutyrate
Biosynthesis and Growth on C1 and C2 Compounds
in the Methylotroph Methylobacterium extorquens
AM1
Department of Chemical Engineering1 and Department of Microbiology,2 University of Washington, Seattle, Washington 98195-1750
Received 3 August 2000/Accepted 7 November 2000
Several DNA regions containing genes involved in
poly-
-hydroxybutyrate (PHB) biosynthesis and degradation and also in
fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB
biosynthesis include those encoding
-ketothiolase
(phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA)
reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the
chromosome together with a third gene with identity to a regulator of
PHB granule-associated protein, referred to as orf3.
phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and
depB). These genes show a high level of identity with each
other at both DNA and amino acid levels. In addition, a gene encoding
-hydroxybutyrate dehydrogenase (hbd) was identified.
Insertion mutations were introduced into depA, depB, phaA, phaB,
phaC, and hbd and also in a gene predicted to encode
crotonase (croA), which is involved in fatty acid
degradation, to investigate their role in PHB cycling. Mutants in
depA, depB, hbd, and croA all produced normal
levels of PHB, and the only growth phenotype observed was the inability
of the hbd mutant to grow on
-hydroxybutyrate. However,
the phaA, phaB, and phaC mutants all showed
defects in PHB synthesis. Surprisingly, these mutants also showed
defects in growth on C1 and C2 compounds and,
for phaB, these defects were rescued by glyoxylate
supplementation. These results suggest that
-hydroxybutyryl-CoA is
an intermediate in the unknown pathway that converts acetyl-CoA to
glyoxylate in methylotrophs and Streptomyces spp.
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