Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16ß), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(ß) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16ß) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16ß) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16ß) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

AIMS: The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. METHODS AND RESULTS: Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. CONCLUSIONS: A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics.

Characterization of Acrylamidase isolated from a newly isolated acrylamide-utilizing bacterium, Ralstonia eutropha AUM-01.

A mesophilic bacterium capable of utilizing acrylamide was isolated, AUM-01, from soil collected from leaf litter at Picnic Point on the UW-Madison campus. In minimal medium with acrylamide as the sole carbon and nitrogen source, a batch culture of AUM-01 completely converted 28.0 mM acrylamide to acrylic acid in 8 h and reached a cell density of 0.3 (A600)). Afterward all the acrylic acid was degraded by 20 h with the cell density increasing to 1.9 (A600). The acrylamide-utilizing bacterium was identified as Ralstonia eutropha based on morphological observations, the BiOLOG GN2 MicroPlate™ identification system for Gram-negative bacteria, and additional physiological tests. An acrylamidase that hydrolyzes acrylamide to acrylic acid was purified from the strain AUM-01. The molecular weight of the enzyme from AUM-01 was determined to be 38 kDa by SDS-PAGE. The enzyme had pH and temperature optima of 6.3 and 55°C, and the influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The enzyme from AUM-01 was totally inhibited by ZnSO4 and AgNO3.

Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16.

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a ß-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.

Elucidation of beta-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression.

Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid ß-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single ß-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.

Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134.

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as a sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that the mnp cluster might have played important roles in MNP degradation. In order to experimentally confirm the prediction, we have now characterized mnpA-encoded meta-nitrophenol nitroreductase involved in the initial reaction of MNP degradation. Real-time PCR analysis indicated that mnpA played an essential role in MNP degradation. MnpA was purified to homogeneity as His-tagged proteins and was considered to be a dimer as determined by gel filtration. MnpA was an MNP nitroreductase with a tightly bound flavin mononucleotide (FMN), catalyzing the partial reduction of MNP to meta-hydroxylaminophenol via meta-nitrosophenol in the presence of NADPH and oxygen. The accumulation of meta-nitrosophenol was confirmed with the results of liquid chromatography-diode array detection and time-of-flight mass spectrometry for the first time. The low K (m) and high k (cat) of MnpA as well as MNP-inducible transcription of mnpA suggested that MNP was the physiological substrate for this nitroreductase. In addition, the phylogenetic analysis revealed that nitroreductases of known physiological function including MnpA constituted a new clade in the nitro-FMN-reductase superfamily.

Genome-wide transcriptome analyses of the 'Knallgas' bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism.

Ralstonia eutropha H16 is probably the best-studied 'Knallgas' bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB(-)4 and Delta phaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB(-)4 and (iv) cells from the transition phase of R. eutropha Delta phaC1 with those from the transition phase of R. eutropha PHB(-)4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB(-)4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB(-)4. In strain Delta phaC1, phaA and phaB1 were highly induced compared with PHB(-)4. Additionally, the results of this study suggest that mutant strain PHB(-)4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB(-)4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB(-)4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16Delta phaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent.

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

Characterization of new isolated Ralstonia eutropha strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production.

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and gamma-hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of gamma-hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments.

Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov.

Comparative 16S rDNA sequence analysis indicates that two distinct sublineages, with a sequence dissimilarity of >4 % (bootstrap value, 100 %), exist within the genus RALSTONIA: the Ralstonia eutropha lineage, which comprises Ralstonia basilensis, Ralstonia campinensis, R. eutropha, Ralstonia gilardii, Ralstonia metallidurans, Ralstonia oxalatica, Ralstonia paucula, Ralstonia respiraculi and Ralstonia taiwanensis; and the Ralstonia pickettii lineage, which comprises Ralstonia insidiosa, Ralstonia mannitolilytica, R. pickettii, Ralstonia solanacearum and Ralstonia syzygii comb. nov. (previously Pseudomonas syzygii). This phylogenetic discrimination is supported by phenotypic differences. Members of the R. eutropha lineage have peritrichous flagella, do not produce acids from glucose and are susceptible to colistin, in contrast to members of the R. pickettii lineage, which have one or more polar flagella, produce acid from several carbohydrates and are colistin-resistant. Members of the R. pickettii lineage are viable for up to 6 days on tryptic soy agar at 25 degrees C, whereas members of the R. eutropha lineage are viable for longer than 9 days. It is proposed that species of the R. eutropha lineage should be classified in a novel genus, Wautersia gen. nov. Finally, based on the literature and new DNA-DNA hybridization data, it is proposed that Pseudomonas syzygii should be renamed Ralstonia syzygii comb. nov.

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate.

A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335(T) (DSM 531(T)), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335(T) maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335(T) cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335(T) with this substrate.

Long PCR-amplified rDNA for PCR-RFLP- and Rep-PCR-based approaches to recognize closely related microbial species.

Long PCR was used to amplify a 5-kb fragment of the bacterial ribosomal operon (16S-intergenic spacer region (ISR)-23S) from several Ralstonia eutropha strains (16S rDNA sequence similarity: 97-99%). Due to the large product size, amplicons from the different strains could be distinguished using restriction enzyme fragment length polymorphisms (RFLP) and repetitive PCR analysis (Rep-PCR) with the primer 1492r. These methods may prove useful in differentiating other bacterial strains with highly similar 16S rDNA sequences.

Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

CDC group IV c-2: a new Ralstonia species close to Ralstonia eutropha.

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia.