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

Tombolini, Riccardo; Povolo, Silvana; Buson, Alberto; Squartini, Andrea; Nuti, Marco

doi:10.1099/13500872-141-10-2553

Cited by 58 publications

(45 citation statements)

References 42 publications

(15 reference statements)

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“…Most members of the SDR family of enzymes are NAD(P)-dependent oxidoreductases of 250 to 300 amino acids that contain no metal cofactors and have a wide range of substrate specificities that include alcohols, sugars, prostaglandins, and hydroxysteroids (19,20,34). BadH is most similar to eubacterial acetoacetyl-CoA reductases involved in polyhydroxyalkanoate and fatty acid biosynthesis (about 40% amino acid identity) (36,43). It is 37% identical to 7␣-hydroxysteroid dehydrogenase from E. coli and 36% identical to 20␤-hydroxysteroid dehydrogenase from Streptomyces exfoliatus.…”

Section: Resultsmentioning

confidence: 99%

2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

Pelletier

Harwood

2000

J Bacteriol

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A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD ؉ ; no activity was detected with NADP ؉ as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K m values of 10 and 200 M for 2-hydroxychc-CoA and NAD ؉ , respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate-and cyclohexanecarboxylategrown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.

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Section: Resultsmentioning

confidence: 99%

2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

Pelletier

Harwood

2000

J Bacteriol

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“…The aniA gene is located upstream of phaAB in the opposite orientation (34,46). They demonstrated that aniA is inducible under anaerobic conditions but that the defect of aniA causes little effect on P(3HB) synthesis.…”

Section: Discussionmentioning

confidence: 99%

A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

et al. 2002

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Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chainlength PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions (2,7,20,23). PHAs can be classified into two groups, short chain length (SCL) (3 to 5 carbons per monomer) and medium chain length (MCL) (6 to 14 carbons per monomer) (43). Paracoccus denitrificans, a facultative methylotrophic bacterium, accumulates SCL-PHAs from several alcohols (54). Two gene clusters involved in PHA synthesis and degradation in P. denitrificans have been cloned and characterized. The first is the phaA-phaB gene cluster, which encodes ␤-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase (50), respectively, and the second is the gene cluster phaZ-phaC-phaPphaR, which encodes PHA depolymerase (9), PHA synthase (47), PHA granule-associated phasin (26), and a DNA-binding regulatory protein (24), respectively.Phasins are the most dominant proteins of relatively small molecular size that are associated with PHA granules. It has been proposed that phasins form an amphiphilic layer between the PHA granule and cytoplasm in a manner similar to that of oleosins, which form a layer at the surface of triacylglycerol inclusion in oilseed plants (32,33,42,49). These lipid-bodyassociated proteins exist in several organisms (for a recent review, see reference 30). Phasins also affect the size and the...

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“…(GenBank accession no. U78047), Methylobacterium extorquens (Valentin & Steinbu$ chel, 1993), Paracoccus denitrificans (Ueda et al, 1996), Pseudomonas aeruginosa (Timm & Steinbu$ chel, 1992) (containing two phaC genes), P. oleovorans (Huisman et al, 1991) (containing two phaC genes), Rhizobium etli (Cevallos et al, 1996), Rhizobium meliloti (Tombolini et al, 1995) and Zoogloea ramigera (GenBank accession no. U66242).…”

Section: Methodsmentioning

confidence: 99%

Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR

2000

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Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3 % DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1i 10 5 viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol ; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHApositive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.

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

Cited by 58 publications

References 42 publications

2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR

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