Botho Bowien

Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16

The H2-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H2 and CO2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organisms ability to produce and store large amounts of poly[R-(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

Formation of Enzymes of Autotrophic Metabolism During Heterotrophic Growth of Alcaligenes eutrophus

SUMMARY: Alcaligenes eutrophus strain H16 formed key enzymes of autotrophic metabolism during heterotrophic growth. The formation of the soluble and membrane-bound hydrogenases, ribulose-5-phosphate kinase and ribulosebisphosphate carboxylase were investigated. In addition, selected enzymes shared by autotrophic and heterotrophic carbon metabolism were examined. Key enzymes of autotrophic metabolism were not detected during exponential growth on succinate, pyruvate or acetate, but were found at intermediate activities in cells grown on fructose, gluconate or citrate. Growth with succinate at a suboptimal pH of 7.7 resulted in a decreased growth rate and a marked increase of enzyme activities. Oxygen-limited growth with succinate also led to a derepression of the synthesis of the hydrogenases and the key enzymes of the Calvin cycle. During growth on glycerol or formate, the activities of these enzymes were comparable with those found under autotrophic conditions with H2 and CO2. The results indicate that both the hydrogenases and the key enzymes of the Calvin cycle were formed under conditions of limited availability of energy. Molecular hydrogen was not required for the formation of the hydrogenases. The regulation of the gluconeogenetic enzymes, common to both autotrophic and heterotrophic carbon metabolism, was more balanced. An increase of enzyme activities was observed under autotrophic conditions, in accord with the physiological role of these enzymes under autotrophic and heterotrophic growth conditions.

Genetics and control of CO(2) assimilation in the chemoautotroph Ralstonia eutropha.

Abstract. The nutritional versatility of facultative autotrophs requires efficient overall control of their metabolism. Most of these organisms are Proteobacteria that assimilate CO2 via the highly energy-demanding Calvin-Benson-Bassham reductive pentose-phosphate cycle. The enzymes of the cycle are encoded by cbb genes organized in cbb operons differing in size and composition, although conserved features are apparent. Transcription of the operons, which may form regulons, is strictly controlled, being induced during autotrophic but repressed to varying extents during heterotrophic growth of the bacteria. The chemoautotroph Ralstoniaeutropha is one of the organisms studied extensively for the mechanisms involved in the expression of cbb gene systems. CbbR is a LysR-type transcriptional regulator and the key activator protein of cbb operons. The cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. The activating function of CbbR seems to be modulated by metabolites signaling the nutritional state of the cell to the cbb system. Phosphoenolpyruvate is such a signal metabolite acting as a negative effector of R. eutropha CbbR, whereas NADPH has been proposed to be a coactivator of the protein in two other chemoautotrophs, Xanthobacterflavus and Hydrogenophilusthermoluteolus. There is evidence for the participation of additional regulators in cbb control. In the photoautotrophs Rhodobactercapsulatus and Rhodobactersphaeroides, response regulator RegA of the global two-component signal transduction system RegBA serves this function. It is conceivable that specific variants of cbb control systems have evolved to ensure their optimal integration into regulatory networks operating in the diverse autotrophs characterized by different metabolic capabilities.

Formate and Oxalate Metabolism in Alcaligenes eutrophus

Summary: Alcaligenes eutrophus strain H16 when grown on formate or oxalate as the sole source of carbon and energy had doubling times between 3.5 and 4.5 h. The respective molar growth yields (Y m) were 2.35 and 3.9. During growth on formate or oxalate both a soluble and a membrane-bound formate dehydrogenase were formed. The key enzymes of autotrophic CO2 fixation, ribulose-5-phosphate kinase and ribulosebisphosphate carboxylase, were formed during growth on formate but not on oxalate. Oxalate induced the synthesis of the enzymes of the glycerate pathway. Mutants impaired in autotrophic CO2 fixation but unaffected in the synthesis of the formate dehydrogenases lost their ability to grow on formate but not to grow on oxalate, giving further evidence that formate was assimilated via CO2.

Purification, some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

Abstractd-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2-, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.

Carbonic Anhydrase Is Essential for Growth of Ralstonia eutropha at Ambient CO2 Concentrations

Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO(2)-requiring (HCR) phenotype. Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO(2). In the present study, a gene designated can and encoding a beta-carbonic anhydrase (CA) was identified as the site altered in strain 25-1. The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can. A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1. Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA. The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO(4) for maximal activity. Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO(2) concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO(2) concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified.

STRUCTURAL ANALYSIS OF THE FDS OPERON ENCODING THE NAD+-LINKED FORMATE DEHYDROGENASE OF RALSTONIA EUTROPHA

The fdsGBACD operon encoding the four subunits of the NAD+-reducing formate dehydrogenase ofRalstonia eutropha H16 was cloned and sequenced. Sequence comparisons indicated a high resemblance of FdsA (α-subunit) to the catalytic subunits of formate dehydrogenases containing a molybdenum (or tungsten) cofactor. The NH2-terminal region (residues 1–240) of FdsA, lacking in formate dehydrogenases not linked to NAD(P)+, exhibited considerable similarity to that of NuoG of the NADH:ubiquinone oxidoreductase from Escherichia colias well as to HoxU and the NH2-terminal segment of HndD of NAD(P)+-reducing hydrogenases. FdsB (β-subunit) and FdsG (γ-subunit) are closely related to NuoF and NuoE, respectively, as well as to HoxF and HndA. It is proposed that the NH2-terminal domain of FdsA together with FdsB and FdsG constitute a functional entity corresponding to the NADH dehydrogenase (diaphorase) part of NADH:ubiquinone oxidoreductase and the hydrogenases. No significant similarity to any known protein was observed for FdsD (δ-subunit). The predicted product offdsC showed the highest resemblance to FdhD from E. coli, a protein required for the formation of active formate dehydrogenases in this organism. Transcription of the fdsoperon is subject to formate induction. A promoter structure resembling the consensus sequence of ς70-dependent promoters from E. coli was identified upstream of the transcriptional start site determined by primer extension analysis.

Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus

A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N‐terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7‐7 system. Mobility shift experiments demonstrated that CfxR is a DNA‐binding protein with a target site upstream of both the chromosomal and the plasmid‐encoded cfx operons.

Chromosomally and plasmid-encoded gene clusters for CO2 fixation (cfx genes) in Alcaligenes eutrophus

SummaryTwo sets of structural genes (cfxL, cfxS) for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were localized in the genome of Alcaligenes eutrophus H16 by means of hybridization probes. One set is encoded on a chromosomal 12 kb EcoRI fragment, the other on an 11 kb EcoRI fragment of the megaplasmid pHG1. These fragments have previously been shown to carry the chromosomal and plasmid copies of the also reiterated phosphoribulokinase (PRK) gene (cfxP). Restriction mapping of the cloned fragments showed that, on each of the two genetic entities, the three cfx genes are located in a very similarly organized cluster. The closely linked Rubisco genes are separated from the downstream PRK gene by about 3.8 kb. The two cfx regions are highly homologous as deduced from cross-hybridization experiments. The Rubisco genes were found to be cotranscribed into a 2.1 kb cfxL-cfxS mRNA. Their expression is regulated at the transcriptional level. Rubisco and PRK genes were subcloned and expressed in Escherichia coli under the control of the lac promoter of pUC plasmids. Since active recombinant enzymes with the structural properties of the authentic enzymes were formed, the ribosome binding sites of the genes are recognized in E. coli. A low level of expression of the Rubisco genes occurred even without lac promoter control, indicating some activity of the cfxL-cfxS promoters in the foreign host.

Purification, some catalytic and molecular properties of phosphoribulokinase from Alcaligenes eutrophus.

A key enzyme of the reductive pentose phosphate cycle, phosphoribulokinase (ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was purified from the hydrogen bacterium Alcaligenes eutrophus to apparent homogeneity. The purification procedure involved affinity chromatography on Cibacron Blue-agarose and AMP-agarose as the most effective method. Initial-velocity studies showed that the enzyme has a pH optimum of 8.6. Divalent cations were essential for its activity, with Mg2+ supporting maximal reaction rates. Mn2+, Ca2+, or Co2+ did partially substitute for Mg2+ in the reaction. The enzyme exhibited a high degree of substrate specificity with respect to the sugar phosphate, while the specificity towards the nucleoside triphosphate was less pronounced. Saturation curves for both substrates, ribulose 5-phosphate and ATP, did not follow normal Michaelis-Menten kinetics and the enzyme was activated by NADH. Activation by NADH affected the affinity of the enzyme for its substrates. An apparent activation constant for NADH of Ka = 0.19 mM was obtained. The molecular weight of the native enzyme was determined by sedimentation equilibrium centrifugation to be Mrc=0 = 256 000. Sedimentation velocity studies indicated a sedimentation coefficient of s200, w = 10.9 S. Dissociation and subsequent polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate (SDS) revealed only one type of subunit of molecular weight 33 000. It is concluded that the enzyme is an oligomer consisting of probably eight subunits of identical size.