Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16
© Kaddor and Steinbüchel; licensee Springer. 2011
Received: 23 May 2011
Accepted: 13 July 2011
Published: 13 July 2011
The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed.
Biodiesel (fatty acid methyl esters) is currently beside ethanol the major renewable energy source for substitution of petroleum. During production of biodiesel glycerol occurs as a main by-product (about 10%, w/w), thus saturating the glycerol market. Due to the huge surplus of glycerol that lowers its value, it is important to enlarge the field of its application e.g. as substrate for microbial growth and production of biodegradable polymers which in turn reduces the high production costs of polyhydroxyalkanoates (PHA) in industrial fermentation processes.
Overview of detected and investigated genes involved in PEP-PTS and fructose-specific ABC-type transport in R. eutropha H16
Fructose-specific EIIAMan component
Histidine phosphocarrier protein HPr
Enzyme I component
HPr-related phosphocarrier protein
Nitrogen regulatory EIIANtr component; Mannitol/fructose-specific EIIAMtl component
Phosphocarrier protein, N-Acetylglucosamine-specific EINag-HPrNag-EIIANag components
N-Acetylglucosamine-specific EIIBCNag components
N-Acetylglucosamine-specific outer membrane protein (porin)
Fructose-specific ABC-type transporter, ATPase component
Fructose-specific ABC-type transporter, periplasmic component
Fructose-specific ABC-type transporter, permease component
Growth on glycerol is not linked to the PEP-PTS and occurs very slowly in R. eutropha H16; it leads to strong expression of hydrogenases and enzymes of the CBB cycle, the key components of lithoautotrophic metabolism (Friedrich et al. 1981). Furthermore, gluconeogenetic enzymes as well as increased oxidative stress proteins (ROS) were identified in 2-D gels during growth of R. eutropha on glycerol (Schwartz et al. 2009). The three-carbon non-PTS sugar alcohol glycerol is probably transported across the cytoplasmic membrane through facilitated diffusion mediated by the glycerol uptake facilitator protein GlpF (Sweet et al. 1990; Darbon et al. 1999). Two proteins, a glycerol kinase and a glycerol-3-phosphate dehydrogenase, are involved in the phosphorylation of intracellular glycerol to glycerol 3-phosphate and the subsequent conversion to dihydroxyacetone phosphate (Voegele et al. 1993; Schweizer et al. 1997). The latter is either introduced into gluconeogenesis or catabolized through the ED pathway via pyruvate to acetyl-CoA, the precursor for the TCC and for poly(3-hydroxybutyrate), PHB, biosynthesis. In R. eutropha like in most other bacteria, this polyester serves as storage for carbon and energy. It is synthesized in the cytoplasm via acetoacetyl-CoA and 3-hydroxybutyryl-CoA using enzymes encoded by phaA, phaB and phaC under conditions of carbon overflow and nitrogen limitation (Schlegel et al. 1961a; Schindler 1964; Haywood et al. 1988a, b, 1989). PHB is biodegradable and may replace petroleum-derived polyolefins that are widely used e.g. as packaging materials or in medicine (Solaiman et al. 2006). The cost of carbon substrate in large scale PHA production processes can be as high as 50% of the total operating costs (Lee 2006). Abundant raw glycerol may substitute traditionally used carbohydrates in industrial microbial processes and reduce PHA production costs (Murarka et al. 2008; da Silva et al. 2009). The price for crude glycerol is decreasing continuously and amounts currently to 180-220 € per ton (ICIS pricing 2008). Several laboratories investigated the use of glycerol as fermentation substrate for PHA production in different bacteria, e.g. in Pseudomonas as well as Burkholderia species (Ashby et al. 2004, 2005; Chee et al. 2010; Zhu et al. 2010). Moreover, an attempt to produce PHB by R. eutropha JMP134 and a R. eutropha mutant (DSM 545) using commercial and waste glycerol as carbon source was already performed (Mothes et al. 2007; Cavalheiro et al. 2009). However, concerning R. eutropha strain H16 the use of glycerol as a low-cost substrate for growth and biosynthesis of PHB in combination with the high biotechnological potential of this strain has largely been ignored. The present study describes an extension of our previous study (Kaddor and Steinbüchel 2011). Since we observed the involvement of homologous PEP-PTS proteins in the utilization of non-PTS substrates, the main focus of this article is on the importance of PTS homologous proteins and other proteins involved in the carbohydrate uptake system of R. eutropha H16 on the utilization of the slow-growth substrate glycerol, the conversion to PHB, and its effect on carbon metabolism. Furthermore, the use of glycerol as cheap and abundant carbon source for growth of R. eutropha with respect to industrial applications e.g. the production of biodegradable polyesters from renewable resources will be discussed.
Materials and methods
Bacterial strains, media and cultivation conditions
Bacterial strains and mutants used in this study
Reference or source
Smr strain of the wild type H16
PHB-negative mutant of the wild type H16
ptsM precise deletion gene replacement mutant of strain H16
ptsH precise deletion gene replacement mutant of strain H16
ptsI precise deletion gene replacement mutant of strain H16
ptsHI precise deletion gene replacement mutant of strain H16
ptsMHI precise deletion gene replacement mutant of strain H16
H16_A2203 precise deletion gene replacement mutant of strain H16
ptsH, H16_A2203 precise deletion gene replacement mutant of strain H16
frcACB precise deletion gene replacement mutant of strain H16
ptsMHI, frcACB precise deletion gene replacement mutant of strain H16
nagFEC precise deletion gene replacement mutant of strain H16
ptsMHI, nagFEC precise deletion gene replacement mutant of strain H16
H16_A0384 precise deletion gene replacement mutant of strain H16
ptsMHI, H16_A0384 precise deletion gene replacement mutant of strain H16
strain HF39 with Tn5-inertion in ptsI, Smr Kmr
strain HF39 with Tn5-insertion in ptsM-ptsH, Smr Kmr
Lyophilized cell material of R. eutropha (5-10 mg) was subjected to methanolysis in presence of 85% (v/v) methanol and 15% (v/v) sulfuric acid for 3 h at 100°C. The resulting methyl esters of 3-hydroxybutyrate were characterized by gas chromatography as described previously (Brandl et al. 1988; Timm and Steinbüchel 1990) by using an Agilent 6850 GC (Agilent Technologies, Waldbronn, Germany) equipped with a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE, Darmstadt, Germany) and a flame ionization detector (Agilent Technologies).
Growth behavior of mutants in liquid media containing glycerol
As the most obvious result of these experiments two groups of mutants with different growth and accumulation behavior were revealed. Figure 1 summarizes the results of the cultivation experiments in MSM containing glycerol.
The first group A (Figure 1a) is represented by the wild type H16 whose increase of optical density ceased after 150 h of cultivation and exhibited a maximum optical density of 720 KU (350 h). Mutants H16 ΔH16_A2203, H16 ΔH16_A0384, H16 ΔptsMHI ΔH16_A0384, H16 ΔfrcACB and H16 ΔnagFEC belonging to group A behaved similarily like the wild type.
PHB accumulation of mutants utilizing glycerol as a sole carbon source
In comparison to accumulation experiments made in MSM plus sodium gluconate or fructose (Kaddor and Steinbüchel 2011), the capability of some mutants to accumulate PHB was reduced up to 24% (w/w) of cell dry matter when cells were cultivated in MSM containing glycerol. This may be due to the limited number of available carbon and to the competition of PHB biosynthesis and gluconeogenesis for C3-intermediates required for product formation and growth (Bormann and Roth 1999). As expected, strain H16 synthesized large amounts of PHB (70.7%, w/w) in the early stationary growth phase (95 h), whereas strain PHB-4 did not accumulate any detectable polyester at all. In contrast, Chee et al. (2010) obtained only about 33% (w/w) PHB in the cells during cultivation of the wild type H16 in modified MSM with glycerol as a sole carbon source for 72 h.
Mutant strains belonging to group A stored PHB in the range of 49-71.3% (w/w) at the maximum, whereas in strains belonging to group B PHB contents of 20.1% (w/w) were not exceeded. In this group, the lowest PHB contents were obtained for the Tn5-induced mutant HF39 ptsI::Tn5::mob which seemed not to enhance PHB biosynthesis in the early stationary phase (7.8%, w/w). Moreover, the strain did not degrade any PHB after induction with ammonium chloride although the optical density increased after this time (Figure 1b). Mutant HF39 ptsMH::Tn5::mob behaved similarly to this mutant which implies that the inserted Tn5 affected synthesis as well as mobilization of PHB. This observation was not made when sodium gluconate, fructose or N-acetylglucosamine were used as carbon source (Kaddor and Steinbüchel 2011).
When comparing group A with group B, it is noticeable that the deletion of ptsM, ptsH, or ptsI exerted a significant change of the PHB synthesis phenotype. Besides the Tn5-induced mutants, the remaining mutants of group B harbored in addition the deletion of either ptsM, ptsH, ptsI or all three genes. Another mutant, H16 ΔptsHI behaved similar like mutant H16 ΔptsMHI (data not shown). The impact of the putative ptsMHI operon was observed during growth in presence of both, the PTS carbohydrate N-acetylglucosamine and the non-PTS carbon sources sodium gluconate, fructose, and glycerol. Particularly, during growth on glycerol in comparison to growth on the previously analyzed carbon sources (Kaddor and Steinbüchel 2011), mutants defective in the putative ptsMHI operon accumulated less PHB than the wild type. Pries et al. (1991) made similar observations with Tn5-induced ptsH/ptsI mutants exhibiting a PHB-leaky phenotype with a lower PHB content of the cells when grown on gluconate. However, a faster mobilization of PHB after exhausting the extracellular carbon source, as it occurred in presence of gluconate, was not noticed when cultivated in media containing glycerol. Despite the still unknown functions of ptsH and ptsI, an exclusively regulatory role in PHB and carbon metabolism was already proposed (Pries et al. 1991; Kaddor and Steinbüchel 2011).
Additionally, our study gives evidence for the involvement of ptsM (fructose-specific EIIAMan) in this regulatory mechanism, indicating a functional ptsMHI operon which is supported by the corresponding gene organization. It has already been proven that PtsM is not involved in fructose uptake and transport (Kaddor and Steinbüchel 2011), and therefore, the relation to EIIAMan remained undetermined. Mutant H16 ΔH16_A0384 lacking the nitrogen regulatory EIIANtr component did not show PHB overproduction in MSM plus glycerol (63.6%, w/w, PHB) as it was observed during growth in MSM plus gluconate (87.6%, w/w, PHB). As it is obvious from the quadruple mutant H16 ΔptsMHI ΔH16_A0384 (49%, w/w, PHB), the high decrease of PHB production in the triple mutant H16 ΔptsMHI (17.5%, w/w, PHB) seemed to be compensated by the additional deletion of H16_A0384 that has also been observed during cultivation experiments with the non-PTS sugars sodium gluconate or fructose as carbon source (Kaddor and Steinbüchel 2011). In disruption mutants of Azotobacter vinelandii UW136, RN7 (ptsN::Kmr ptsP::Tcr) and RN8 (ptsN::Kmr ptsO::Spr), the negative effect of the single ptsP or ptsO mutation on PHB accumulation was suppressed in the double mutants as well (Noguez et al. 2008). The same result was obtained for a Pseudomonas putida MAD2 double mutant (ptsN::xylE ptsO::Kmr) (Velázquez et al. 2007). Unlike the mutation of ΔH16_A0384, the deletion of H16_A2203 (HPr-related phosphocarrier protein), frcACB (fructose-specific ABC-type transporter) or nagFEC (PTSNag) could not enhance the growth and limited PHB accumulation of the derived multiple mutants H16 ΔptsH ΔH16_A2203, H16 ΔptsMHI ΔfrcACB and H16 ΔptsMHI ΔnagFEC.
The limited carbohydrate utilization range of R. eutropha coupled with the high costs of these carbon sources in biopolymer production restricts its application in biotechnological processes. Renewable substitutes for the so far used expensive substrates must be investigated to lower the commercial PHA production costs (e.g. of the thermoplastic Biopol) to make them competitive with the petrochemical-based plastic manufacture. Based on the experimental results, it appears that polymer accumulation in strain H16 is reduced to a minor extend when cells were grown on glycerol (70.7%, w/w, PHB after 260 h of cultivation) in comparison to accumulation experiments made on sodium gluconate or fructose (up to 78%, w/w, PHB after 28 h of cultivation, Kaddor and Steinbüchel 2011). We demonstrated that strain H16 has the potential to utilize glycerol for indeed suboptimal growth but with an unrestricted capability of valuable PHB production. Although glycerol transport and utilization is independent of the PEP-PTS in strain H16, deletion of the PTS homologous genes affected anyhow carbon and PHB metabolism in this strain indicating a complex regulatory function of the PTS. However, the slow growth of the wild type on this cheap and abundant sugar substitute prevents it currently from its use in industrial large-scale productions. Heterotrophic growth on glycerol is related to carbon and energy limiting conditions. Besides oxidative stress proteins and hydrogenases, enzymes of gluconeogenesis and the CBB pathway as well as PhaA and PhaB belong to the most abundant proteins of glycerol-grown cells (Friedrich et al. 1981; Schwartz et al. 2009).
This study focussed on the involvement of PTS homologous proteins on the utilization of glycerol with respect to polymer biosynthesis in R. eutropha H16. Four PTS homologous proteins (PtsM, PtsH, PtsI, PtsN) showed a significant influence during glycerol utilization on both, cell growth and PHB accumulation. Deletion of the fructose-specific transport proteins resulted in no significant difference to the wild type concerning growth and storage behavior. Due to the occurrence of PEP-PTS homologous proteins and the absence of a PTS-mediated carbohydrate uptake in this strain except for the PTSNag, further investigations are required to unravel their functions in this PHB producing strain. Besides the generation of deletion mutants and their phenotypical characterization, intensive studies on the putative operon ptsMHI are now necessary to characterize the respective genes in more detail and to resolve their roles in the metabolism of R. eutropha. Certainly, this study provides a further degree of regulation between the general PTS proteins and both, PHB and carbon metabolism in R. eutropha H16.
This study was financially supported by the Bundesministerium für Bildung und Forschung (BMBF, FKZ-0313751) within the Competence Network Göttingen "Genome Research on Bacteria". We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publication Fund of University of Muenster.
- Ashby RD, Solaiman DKY, Foglia TA: Bacterial poly(hydroxyalkanoate) polymer production from the biodiesel co-product stream. J Polym Environ 2004, 12: 105–112.View ArticleGoogle Scholar
- Ashby RD, Solaiman DKY, Foglia TA: Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol. Biomacromolecules 2005, 6: 2106–2112. 10.1021/bm058005hPubMedView ArticleGoogle Scholar
- Barabote RD, Saier MH: Comparative genomic analyses of the bacterial phosphotransferase system. Microbiol Mol Biol Rev 2005, 69: 608–634. 10.1128/MMBR.69.4.608-634.2005PubMed CentralPubMedView ArticleGoogle Scholar
- Bormann EJ, Roth M: The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates. Biotechnol Lett 1999, 21: 1059–1063. 10.1023/A:1005640712329View ArticleGoogle Scholar
- Brandl H, Gross RA, Lenz RW, Fuller RC: Pseudomonas oleovorans as a source of poly(β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 1988, 66: 2117–2124.Google Scholar
- Cases I, Velázquez F, de Lorenzo V: The ancestral role of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as exposed by comparative genomics. Res Microbiol 2007, 158: 666–670. 10.1016/j.resmic.2007.08.002PubMedView ArticleGoogle Scholar
- Cavalheiro JMBT, de Almeida MCMD, Grandfils C, da Fonseca MMR: Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process Biochem 2009, 44: 509–515. 10.1016/j.procbio.2009.01.008View ArticleGoogle Scholar
- Chee JY, Tan Y, Samian MR, Sudesh K: Isolation and characterization of a Burkholderia sp. USM (JCM15050) capable of producing polyhydroxyalkanoate (PHA) from triglycerides, fatty acids and glycerols. J Polym Environ 2010, 18: 584–592. 10.1007/s10924-010-0204-1View ArticleGoogle Scholar
- Commichau FM, Forchhammer K, Stülke J: Regulatory links between carbon and nitrogen metabolism. Curr Opin Microbiol 2006, 9: 167–172. 10.1016/j.mib.2006.01.001PubMedView ArticleGoogle Scholar
- da Silva GP, Mack M, Contiero J: Glycerol: A promising and abundant carbon source for industrial microbiology. Biotechnol Adv 2009, 27: 30–39. 10.1016/j.biotechadv.2008.07.006PubMedView ArticleGoogle Scholar
- Darbon E, Ito K, Huang HS, Yoshimoto T, Poncet S, Deutscher J: Glycerol transport and phophoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus . Microbiology 1999, 145: 3205–3212.PubMedGoogle Scholar
- Deutscher J, Francke C, Postma PW: How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 2006, 70: 939–1031. 10.1128/MMBR.00024-06PubMed CentralPubMedView ArticleGoogle Scholar
- Friedrich CG, Friedrich B, Bowien B: Formation of enzymes of autotrophic metabolism during heterotrophic growth of Alcaligenes eutrophus . J Gen Microbiol 1981, 122: 69–78.Google Scholar
- Gottschalk G, Eberhardt U, Schlegel HG: Verwertung von Fructose durch Hydrogenomonas H16 (I.). Arch Mikrobiol 1964, 48: 95–108. 10.1007/BF00406600PubMedView ArticleGoogle Scholar
- Haywood GW, Anderson AJ, Chu L, Dawes EA: Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus . FEMS Microbiol Lett 1988, 52: 91–96. 10.1111/j.1574-6968.1988.tb02577.xView ArticleGoogle Scholar
- Haywood GW, Anderson AJ, Chu L, Dawes EA: The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus . FEMS Microbiol Lett 1988, 52: 259–264. 10.1111/j.1574-6968.1988.tb02607.xView ArticleGoogle Scholar
- Haywood GW, Anderson AJ, Dawes EA: The importance of PHB-synthase substrate specificity in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus . FEMS Microbiol Lett 1989, 57: 1–6. 10.1111/j.1574-6968.1989.tb03210.xView ArticleGoogle Scholar
- ICIS pricing: Reed Business Information Limited. 2008. [http://www.icispricing.com/]Google Scholar
- Kaddor C, Steinbüchel A: Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16. Appl Environ Microbiol 2011, 77: 3582–3590. 10.1128/AEM.00218-11PubMed CentralPubMedView ArticleGoogle Scholar
- Kanehisa M, Goto S, Kawashima S, Nakaya A: The KEGG database at GenomeNet. Nucleic Acids Res 2002, 30: 42–46. 10.1093/nar/30.1.42PubMed CentralPubMedView ArticleGoogle Scholar
- Kotrba P, Inui M, Yukawa H: Bacterial phosphotransferase system (PTS) in carbohydrate uptake and control of carbon metabolism. J Biosci Bioeng 2001, 92: 502–517. 10.1263/jbb.92.502PubMedView ArticleGoogle Scholar
- Krauße D, Hunold K, Kusian B, Lenz O, Stülke J, Bowien B, Deutscher J: Essential role of the hprK gene in Ralstonia eutropha H16. J Mol Microbiol Biotechnol 2009, 17: 146–152. 10.1159/000233505PubMedView ArticleGoogle Scholar
- Lee SY: Deciphering bioplastic production. Nat Biotechnol 2006, 24: 1227–1229. 10.1038/nbt1006-1227PubMedView ArticleGoogle Scholar
- Mothes G, Schnorpfeil C, Ackermann JU: Production of PHB from crude glycerol. Eng Life Sci 2007, 7: 475–479. 10.1002/elsc.200620210View ArticleGoogle Scholar
- Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R: Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 2008, 74: 1124–1135. 10.1128/AEM.02192-07PubMed CentralPubMedView ArticleGoogle Scholar
- Noguez R, Segura D, Moreno S, Hernandez A, Juarez K, Espín G: Enzyme I Ntr , NPr and IIA Ntr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii . J Mol Microbiol Biotechnol 2008, 15: 244–254. 10.1159/000108658PubMedView ArticleGoogle Scholar
- Pflüger K, de Lorenzo V: Evidence of in vivo cross talk between the nitrogen-related and fructose-related branches of the carbohydrate phosphotransferase system of Pseudomonas putida . J Bacteriol 2008, 190: 3374–3380. 10.1128/JB.02002-07PubMed CentralPubMedView ArticleGoogle Scholar
- Pflüger-Grau K, Görke B: Regulatory roles of the bacterial nitrogen-related phosphotransferase system. Trends Microbiol 2010, 18: 205–214. 10.1016/j.tim.2010.02.003PubMedView ArticleGoogle Scholar
- Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Pötter M, Schwartz E, Strittmatter A, Voß I, Gottschalk G, Steinbüchel A, Friedrich B, Bowien B: Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat Biotechnol 2006, 24: 1257–1262. 10.1038/nbt1244PubMedView ArticleGoogle Scholar
- Pries A, Priefert H, Krüger N, Steinbüchel A: Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the phenotype poly(β-hydroxybutyric acid)-leaky which exhibit homology to ptsH and ptsI of Escherichia coli . J Bacteriol 1991, 173: 5843–5853.PubMed CentralPubMedGoogle Scholar
- Reizer J, Reizer A, Saier MH Jr, Jacobson GR: A proposed link between nitrogen and carbon metabolism involving protein phosphorylation in bacteria. Protein Sci 1992, 1: 722–726. 10.1002/pro.5560010604PubMed CentralPubMedView ArticleGoogle Scholar
- Schindler J: Die Synthese von Poly-β-hydroxybuttersäure durch Hydrogenomonas H16: Die zu β-Hydroxybutyryl-Coenzym A führenden Reaktionsschritte. Arch Mikrobiol 1964, 49: 236–255. 10.1007/BF00409747PubMedView ArticleGoogle Scholar
- Schlegel HG, Gottschalk G, Bartha V: Formation and utilization of poly-β-hydroxybutyric acid by knallgas bacteria ( Hydrogenomonas ). Nature 1961, 29: 463–465.View ArticleGoogle Scholar
- Schlegel HG, Kaltwasser H, Gottschalk G: Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 1961, 38: 209–222. 10.1007/BF00422356PubMedView ArticleGoogle Scholar
- Schubert P, Steinbüchel A, Schlegel HG: Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli . J Bacteriol 1988, 170: 5837–5847.PubMed CentralPubMedGoogle Scholar
- Schwartz E, Henne A, Cramm R, Eitinger T, Friedrich B, Gottschalk G: Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H 2 -based lithoautotrophy and anaerobiosis. J Mol Biol 2003, 332: 369–383. 10.1016/S0022-2836(03)00894-5PubMedView ArticleGoogle Scholar
- Schwartz E, Voigt B, Zühlke D, Pohlmann A, Lenz O, Albrecht D, Schwarze A, Kohlmann Y, Krause C, Hecker M, Friedrich B: A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16. Proteomics 2009, 9: 5132–5142. 10.1002/pmic.200900333PubMedView ArticleGoogle Scholar
- Schweizer HP, Jump R, Po C: Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator ( glpF ) and glycerol kinase ( glpK ) of Pseudomonas aeruginosa . Microbiology 1997, 143: 1287–1297. 10.1099/00221287-143-4-1287PubMedView ArticleGoogle Scholar
- Solaiman DKY, Ashby RD, Foglia TA, Marmer WN: Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Appl Microbiol Biotechnol 2006, 71: 783–789. 10.1007/s00253-006-0451-1PubMedView ArticleGoogle Scholar
- Srivastava S, Urban M, Friedrich B: Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn 5 . Arch Microbiol 1982, 131: 203–207. 10.1007/BF00405879PubMedView ArticleGoogle Scholar
- Stülke J, Hillen W: Coupling physiology and gene regulation in bacteria: the phosphotransferase sugar uptake system delivers the signals. Naturwissenschaften 1998, 85: 583–592. 10.1007/s001140050555PubMedView ArticleGoogle Scholar
- Sweet G, Gandor C, Voegele R, Wittekindt N, Beuerle J, Truniger V, Lin ECC, Boos W: Glycerol facilitator of Escherichia coli : cloning of glpF and identification of the glpF product. J Bacteriol 1990, 172: 424–430.PubMed CentralPubMedGoogle Scholar
- Timm A, Steinbüchel A: Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol 1990, 56: 3360–3367.PubMed CentralPubMedGoogle Scholar
- Velázquez F, Pflüger K, Cases I, De Eugenio LI, de Lorenzo V: The phosphotransferase system formed by PtsP, PtsO, and PtsN proteins controls production of polyhydroxyalkanoates in Pseudomonas putida . J Bacteriol 2007, 189: 4529–4533. 10.1128/JB.00033-07PubMed CentralPubMedView ArticleGoogle Scholar
- Voegele RT, Sweet GD, Boos W: Glycerol kinase of Escherichia coli is activated by interaction with glycerol facilitator. J Bacteriol 1993, 175: 1087–1094.PubMed CentralPubMedGoogle Scholar
- Zhu C, Nomura CT, Perrotta JA, Stipanovic AJ, Nakas JP: Production and characterization of poly-3-hydroxybutyrate from biodiesel-glycerol by Burkholderia cepacia ATCC 17759. Biotechnol Prog 2010, 26: 424–430.PubMedGoogle Scholar
- Zimmer B, Hillmann A, Görke B: Requirements for the phosphorylation of the Escherichia coli EIIA Ntr protein in vivo . FEMS Microbiol Lett 2008, 286: 96–102. 10.1111/j.1574-6968.2008.01262.xPubMedView ArticleGoogle Scholar
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