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1.  Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16 
AMB Express  2014;4:2.
Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium’s capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle. Analysis of the R. eutropha H16 genome sequence revealed the presence of four CA genes: can, can2, caa and cag. We evaluated the importance of each of the CAs in the metabolism of R. eutropha by examination of growth and enzyme activity in gene deletion, complementation, and overexpression strains. All four purified CAs were capable of performing the interconversion of CO2 and HCO3–, although the equilibrium towards the formation of CO2 or HCO3– differs with each CA. Deletion of can, encoding a β-CA, affected the growth of R. eutropha; however the growth defect could be compensated by adding CO2 to the culture. Deletion of the caa, encoding an α-CA, had the strongest deleterious influence on cell growth. Strains with deletion or overexpression of can2 or cag genes exhibited similar behavior to wild type under most of the conditions tested. In this work, Caa was studied in greater detail using microscopy and complementation experiments, which helped confirm its periplasmic localization and determine its importance for robust growth of R. eutropha. A hypothesis for the coordinated role of these four enzymes in the metabolism of R. eutropha is proposed.
doi:10.1186/2191-0855-4-2
PMCID: PMC3904209  PMID: 24410804
CO2 transport; Cupriavidus necator; Carbon dioxide; Bicarbonate; Dissolved inorganic carbon; Periplasm; Zinc metalloenzyme
2.  Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16 
Applied and Environmental Microbiology  2012;78(22):8033-8044.
Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor σ54 increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with dl-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.
doi:10.1128/AEM.01693-12
PMCID: PMC3485964  PMID: 22961894
3.  Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications in the initiation and elongation of polymer formation in vivo 
Biochemistry  2012;51(11):2276-2288.
Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaCEc), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaCRe) was constructed and integrated into the R. eutropha genome in place of the wt-phaC. Strep2-PhaCRe was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cdw) and 24 h (PHB, 2% cdw). Analysis of the purified PhaC by size exclusion chromatography, SDS-PAGE and gel permeation chromatography revealed that it unexpectedly co-purified with the phasin protein, PhaP1, and with soluble PHB (Mw 350 kDa) in a “high molecular weight” (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays to monitor PHB formation in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaCRe (and PhaCEc), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and non-primed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.
doi:10.1021/bi2013596
PMCID: PMC3326396  PMID: 22369488
Ralstonia eutropha; native and primed PHB synthase; Strep2 tag; soluble granules
4.  Examination of PHB Depolymerases in Ralstonia eutropha: Further Elucidation of the Roles of Enzymes in PHB Homeostasis 
AMB Express  2012;2:26.
Polyhydroxyalkanoates (PHA) are biodegradable polymers that are attractive materials for use in tissue engineering and medical device manufacturing. Ralstonia eutropha is regarded as the model organism for PHA biosynthesis. We examined the effects of PHA depolymerase (PhaZ) expression on PHA homeostasis in R. eutropha strains. In order to analyze the impact of PhaZs on R. eutropha granule architecture, we performed electron microscopy on several phaZ knockout strains and the wild type strain grown under PHA production conditions. Analysis of the acquired micrographs was based on stereology: the ratio of granule area and cell area was determined, along with total granule count per full-size cell image. Cells bearing a phaZ2 knockout mutation alone or in conjunction with a phaZ1 mutation were found to have a high granule volume per cell volume and a higher granule count compared to wild type. A phaZ quadruple knockout strain appeared to have a low granule volume per cell volume and a low granule count per cell. Cells bearing a phaZ3 knockout were found to have a higher granule count than the wild type, whereas granule volume per cell volume was similar. Accordingly, we hypothesize that PhaZs have not only an impact on PHA degradation but also on the 3-dimensional granule architecture. Based on our data, PhaZ2 is postulated to affect granule density. This work increased our knowledge about PHA depolymerases in R. eutropha, including enzymes that had previously been uncharacterized.
doi:10.1186/2191-0855-2-26
PMCID: PMC3430594  PMID: 22537946
Ralstonia eutropha; Polyhydroxyalkanoates; Polyhydroxybutyrate; Biomaterials; Depolymerase; Granules; Carbon utilization; Electron microscopy; Stereology
5.  Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2▿ 
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaCCs). PhaCCs showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaCCs expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCs was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaCCs of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCs is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
doi:10.1128/AEM.01997-10
PMCID: PMC3126384  PMID: 21398494
6.  Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression▿ †  
Journal of Bacteriology  2010;192(20):5454-5464.
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 β-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 β-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.
doi:10.1128/JB.00493-10
PMCID: PMC2950501  PMID: 20709892
7.  Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from Bacteroides fragilis and Their Role in Hexose and N-Acetylglucosamine Utilization 
Journal of Bacteriology  2005;187(3):890-901.
Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-d-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.
doi:10.1128/JB.187.3.890-901.2005
PMCID: PMC545704  PMID: 15659667

Results 1-7 (7)