Identification of a lipoamide dehydrogenase gene as second locus affected in poly(3-hydroxybutyric acid)-leaky mutants of Alcaligenes eutrophus

Pries, Andreas; Hein, Silke; Steinbüchel, Alexander

doi:10.1016/0378-1097(92)90341-k

Cited by 7 publications

(13 citation statements)

References 28 publications

(14 reference statements)

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“…The in silico analysis also confirmed the presence of a genomic EcoRI fragment comprising all three genes of the PDHC, in agreement with the 6.3-kbp EcoRI fragment, which restored the wild-type phenotype in the PHB-leaky Tn5 mutant (46).…”

Section: Resultssupporting

confidence: 73%

“…The published data for the PHB-leaky Tn5 mutants exhibiting a reduced PHB accumulation rate (21,46) were verified and completed. Our investigations revealed that mutants H1474 and H1479 are most probably siblings of mutant H1482 because the Tn5 insertions had occurred at identical positions.…”

Section: Resultsmentioning

confidence: 88%

“…Pries et al reported in 1992 on a Tn5-induced PHB-leaky mutant (H1482) showing reduced PHB accumulation (46). This mutant grew similarly to the wild type; however, the DHLDH activity was significantly lower, exhibiting only 29% of the activity of the wild type.…”

mentioning

confidence: 99%

“…The H1482 mutant, as well as the other PHB-leaky subclass B mutants, H1474, H1479, and H1485, could be complemented by plasmid pVK6300, which harbored a 6.3-kbp EcoRI restriction fragment of the wild type. Sequence analysis mapped the Tn5 insertion in the pdhL gene (46). On the protein level, the insertion locus corresponded to the carboxyl terminus of the DHLDH, which is part of the PDHC (21,46).…”

mentioning

confidence: 99%

“…Sequence analysis mapped the Tn5 insertion in the pdhL gene (46). On the protein level, the insertion locus corresponded to the carboxyl terminus of the DHLDH, which is part of the PDHC (21,46). The available genome sequence of R. eutropha H16 (43) encouraged the elucidation of this interesting phenomenon and an attempt to unravel the unknown linkage between phenotype and genotype.…”

mentioning

confidence: 99%

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Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

Raberg

Bechmann

Brandt

et al. 2011

Appl Environ Microbiol

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A previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutant Ralstonia eutropha H1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-type R. eutropha H16 but similar growth behavior. Insertion of Tn5 was localized in the pdhL gene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence of R. eutropha H16, observations were verified and further detailed analyses and experiments were done. In silico genome analysis revealed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5 in pdhL of mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ⌬pdhL deletion mutant of R. eutropha exhibited the same phenotype as the Tn5 mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5 mutant H1482. However, insertion of Tn5 or deletion of pdhL decreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components of R. eutropha H16 or of other bacteria, like Burkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component of B. cepacia possesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to the loss of PdhL on the cellular level.Ralstonia eutropha H16 is a Gram-negative, rod-shaped, and facultative chemolithoautotrophic hydrogen-oxidizing bacterium and has served as a model organism for polyhydroxyalkanoate (PHA) metabolism and hydrogen-based chemolithoautotrophy for nearly 50 years (12, 51). PHAs are accumulated as granules in the cytoplasm and serve the cells as storage compounds for carbon and energy. PHAs are synthesized under unbalanced growth conditions if a carbon source is present in excess and if another macroelement (N, O, P, or S) is depleted at the same time (3, 63). PHAs are synthesized and accumulated by a large variety of prokaryotes and may represent the major cell constituent, contributing up to about 90% of the cell dry weight (4). Although R. eutropha H16 is able to synthesize different PHAs with short carbon chain lengths (65), poly(3-hydroxybutyric acid) (PHB) is usually the predominant PHA in the bacterium (15,28). When the limiting macroelement that caused PHB accumulation is supplied again, degradation (mobilization) of PHB is induced, and the storage compound is used as a carbon and energy source (67).In addition to the inte...

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

Raberg

Bechmann

Brandt

et al. 2011

Appl Environ Microbiol

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Analysis of the PHA granule‐associated proteins GA20 .and GA11 in Methylobacterium extorquens and Methylobacterium rhodesianum

Föllner

Madkour

Mayer

et al. 1997

J. Basic Microbiol.

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Electrophoretic analysis of the proteins bound to poly(3-hydroxybutyric acid), PHB-, granules in Methylobacterium extorquens, M. rhodesianum as well as the PHB-leaky mutants Mu 1 and Mu 11, which were isolated from the latter, resulted in two dominant low-molecular weight proteins, which were referred to as GA11 and GA20. After purification of these proteins antibodies against the GA11 and GA20 protein of M. extorquens were obtained. Both proteins bound to the surface of PHB granules as revealed by immunoelectron microscopy of whole cells of M. extorquens and M. rhodesianum. With cells of the PHB-leaky mutants Mu 1 and Mu 11 no specific labeling was observed. The N-terminal amino acid sequences of the GA11 and the GA20 protein were determined. We found significant homologies between the sequences of the investigated strains. The use of oligonucleotide probes based on the N-terminal sequences of the GA20 protein from M. rhodesianum to identify the corresponding structural genes in various genomic libraries failed.

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Poly- -hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41

et al. 1995

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Genes encoding fiketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORFl were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB-Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB-strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORFl with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

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Identification of a lipoamide dehydrogenase gene as second locus affected in poly(3-hydroxybutyric acid)-leaky mutants of Alcaligenes eutrophus

Cited by 7 publications

References 28 publications

Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

Analysis of the PHA granule‐associated proteins GA20 .and GA11 in Methylobacterium extorquens and Methylobacterium rhodesianum

Poly- -hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41

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