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1.  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.
PMCID: PMC3485964  PMID: 22961894
2.  Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors 
Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. A significant technical challenge to using lignocellulose is the presence of microbial growth inhibitors generated during pretreatment processes. Triacylglycerols (TAGs) are potential precursors for lipid-based biofuel production. Rhodococcus opacus MITXM-61 is an oleaginous bacterium capable of producing large amounts of TAGs on high concentrations of glucose and xylose present in lignocellulosic hydrolysates. However, this strain is sensitive to ligonocellulose-derived inhibitors. To understand the toxic effects of the inhibitors in lignocellulosic hydrolysates, strain MITXM-61 was examined for tolerance toward the potential inhibitors and was subjected to adaptive evolution for the resistance to the inhibitors.
We investigated growth-inhibitory effects by potential lignocellulose-derived inhibitors of phenols (lignin, vanillin, 4-hydroxybenzaldehyde (4-HB), syringaldehyde), furans (furfural and 5-hydroxymethyl-2-furaldehyde), and organic acids (levulinic acid, formic acid, and acetic acid) on the growth and TAG production of strain MITXM-61. Phenols and furans exhibited potent inhibitory effects at a concentration of 1 g L−1, while organic acids had insignificant impacts at concentrations of up to 2 g L−1. In an attempt to improve the inhibitor tolerance of strain MITXM-61, we evaluated the adaptation of this strain to the potential inhibitors. Adapted mutants were generated on defined agar media containing lignin, 4-HB, and syringaldehyde. Strain MITXM-61SHL33 with improved multiple resistance of lignin, 4-HB, and syringaldehyde was constructed through adaptive evolution-based strategies. The evolved strain exhibited a two- to threefold increase in resistance to lignin, 4-HB, and syringaldehyde at 50% growth-inhibitory concentrations, compared to the parental strain. When grown in genuine lignocellulosic hydrolysates of corn stover, wheat straw, and hardwood containing growth inhibitors, strain MITXM-61SHL33 exhibited a markedly shortened lag phase in comparison with that of strain MITXM-61.
This study provides important clues to overcome the negative effects of inhibitors in lignocellulosic hydrolysates on TAG production of R. opacus cells. The findings can contribute to significant progress in detoxified pretreatment of hydrolysates and development of more efficient strains for industrial TAG fermentations of R. opacus using lignocellulosic biomass.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0258-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4456722  PMID: 26052344
Triacylglycerol; Rhodococcus opacus; Lignocellulose-derived inhibitor; Tolerance; Adaptation; Lignocellulosic fuel; Lipid-based biofuel
3.  Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels 
Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations.
An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L−1 glycerol, produced 2.3 g L−1 of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g−1 of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L−1) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L−1 glycerol, 40 g L−1 glucose, and 40 g L−1 xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L−1 of TAGs, representing 51.2% of the CDM. In addition, when 20 g L−1 glycerol was pulse-loaded into the culture with 40 g L−1 glucose and 40 g L−1 xylose at the stationary growth phase, MITGM-173 produced 14.3 g L−1 of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L−1 glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g−1 on the initial addition and 0.279 g g−1 on the pulse addition of glycerol.
We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0209-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4355421  PMID: 25763105
Triacylglycerol; Renewable fuels; Rhodococcus opacus; Adaptive evolution; Glycerol utilization; Co-fermentation
4.  Molecular MRI of Collagen to Diagnose and Stage Liver Fibrosis 
Journal of hepatology  2013;59(5):992-998.
Background & Aims
The gold standard in assessing liver fibrosis is biopsy despite limitations like invasiveness and sampling error and complications including morbidity and mortality. Therefore, there is a major unmet medical need to quantify fibrosis noninvasively to facilitate early diagnosis of chronic liver disease and provide a means to monitor disease progression. The goal of this study was to evaluate the ability of several magnetic resonance imaging (MRI) techniques to stage liver fibrosis.
A gadolinium (Gd)-based MRI probe targeted to type I collagen (termed EP-3533) was utilized to noninvasively stage liver fibrosis in a carbon tetrachloride (CCl4) mouse model and the results were compared to other MRI techniques including relaxation times, diffusion and magnetization transfer measurements.
The most sensitive MR biomarker was the change in liver:muscle contrast to noise ratio (ΔCNR) after EP-3533 injection. We observed a strong positive linear correlation between ΔCNR and liver hydroxyproline (i.e. collagen) levels (r=0.89) as well as ΔCNR and conventional Ishak fibrosis scoring. In addition, the area under the receiver operating curve (AUR0C) for distinguishing early (Ishak ≤3) from late (Ishak ≥ 4) fibrosis was 0.942±0.052 (p<0.001). By comparison, other MRI techniques were not as sensitive to changes in fibrosis in this model.
We have developed a MRI technique using a collagen-specific probe for diagnosing and staging liver fibrosis, and validated it in the CCl4 mouse model. This approach should provide a better means to monitor disease progression in patients.
PMCID: PMC3805694  PMID: 23838178
fibrosis; MRI; molecular imaging; type 1 collagen; noninvasive; gadolinium; EP-3533; CCl4
5.  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.
PMCID: PMC3904209  PMID: 24410804
CO2 transport; Cupriavidus necator; Carbon dioxide; Bicarbonate; Dissolved inorganic carbon; Periplasm; Zinc metalloenzyme
6.  The Rhodococcus opacus TadD protein mediates triacylglycerol metabolism by regulating intracellular NAD(P)H pools 
The Gram-positive actinomycete Rhodococcus opacus is widely studied for its innate ability to store large amounts of carbon in the form of triacylglycerol (TAG). Several groups have demonstrated that R. opacus PD630 is capable of storing anywhere from 50 to 76% of its cell dry weight as TAG. While numerous studies have focused on phenomenological aspects of this process, few have sought to identify the underlying molecular and biochemical mechanisms responsible for the biosynthesis and storage of this molecule.
Herein we further our previous efforts to illuminate the black box that is lipid metabolism in actinomycetes using a genetic approach. Utilizing a simple, colorimetric genetic screen, we have identified a gene, referred to herein as tadD (triacylglycerol accumulation deficient), which is critical for TAG biosynthesis in R. opacus PD630. Furthermore, we demonstrate that the purified protein product of this gene is capable of oxidizing glyceraldehyde-3-phosphate, while simultaneously reducing NAD(P)+ to NAD(P)H. Supporting this biochemical data, we observed that the ratio of NAD(P)H to NAD(P)+ is elevated in wildtype cultures grown under lipid production conditions as compared to cultures grown under vegetative growth conditions, while the mutant strain demonstrated no change irrespective of growth conditions. Finally, we demonstrate that over-expressing a putative phosphorylative glyceraldehyde-3-phosphate dehydrogenase leads to decreased TAG production during growth on TAG accumulation conditions.
Taken together, the data support the identification of a key metabolic branch point separating vegetative growth and lipid accumulation lifestyles in Rhodococcus.
PMCID: PMC3827869  PMID: 24209886
Rhodococcus opacus PD630; GapN; triacylglycerol
7.  Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production 
There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose.
We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations.
We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates that the engineered strain possesses a key trait of converters for lipid-based fuels production from lignocellulosic biomass.
PMCID: PMC3848844  PMID: 24041310
Rhodococcus opacus; Streptomyces padanus; Triacylglycerol; Lipid-based biofuel; Lignocellulosic fuel; Lignocellulosic biomass; Xylose; High-cell-density fermentation; xylA; xylB
8.  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.
PMCID: PMC3326396  PMID: 22369488
Ralstonia eutropha; native and primed PHB synthase; Strep2 tag; soluble granules
9.  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.
PMCID: PMC3430594  PMID: 22537946
Ralstonia eutropha; Polyhydroxyalkanoates; Polyhydroxybutyrate; Biomaterials; Depolymerase; Granules; Carbon utilization; Electron microscopy; Stereology
10.  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.
PMCID: PMC3126384  PMID: 21398494
11.  Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains▿† 
The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.
PMCID: PMC3126409  PMID: 21398488
12.  Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development 
PLoS Genetics  2011;7(9):e1002219.
The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.
Author Summary
Biofuels research is focused on understanding the energy-related metabolic capabilities of a broad range of biological species. To this end we sequenced the genome of Rhodococcus opacus PD630, a bacterium that accumulates close to 80% of its cellular dry weight in oil, a rare trait in the prokaryotic and eukaryotic kingdoms. R. opacus PD630 has a large 9.27 Mb genome that contains many homologous genes dedicated to lipid metabolism. The number and novelty of these predicted genes presents a challenge to the complete and accurate metabolic reconstruction of this species' metabolism based only on genome sequence. To refine our sequence-based metabolic reconstruction, we developed a multidisciplinary approach that included integrating the identification of abundant yet uncommon straight-chain odd-carbon lipid biosynthesis and the results of a catabolic screen for growth substrates. Comparative analysis of the R. opacus PD630 genome sequence with those of a group of related species provided a view into how this bacterium became such a remarkable TAGs producer and led to the identification of a set of biofuels target genes for this group of bacteria. Our synthesis of genome sequence and phenotypic information supports a model for the genetic basis for prokaryotic oleaginy and provides key insights for the engineering of next-generation biofuels with genes that are conserved in both prokaryotic and eukaryotic kingdoms.
PMCID: PMC3169528  PMID: 21931557
13.  Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16 ▿ †  
Journal of Bacteriology  2010;192(20):5319-5328.
The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-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.
PMCID: PMC2950492  PMID: 20729355
14.  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.
PMCID: PMC2950501  PMID: 20709892
15.  Analysis of Transient Polyhydroxybutyrate Production in Wautersia eutropha H16 by Quantitative Western Analysis and Transmission Electron Microscopy 
Journal of Bacteriology  2005;187(11):3825-3832.
Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1a, PhaZ1b, and PhaZ1c (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1a were present simultaneously. No PhaZ1b or PhaZ1c was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1a. The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (106 Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was ∼60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and ΔphaR strains in TSB provided further support for an alternative mechanism of granule formation (Tian et al., J. Bacteriol. 187:3814-3824, 2005).
PMCID: PMC1112050  PMID: 15901707
16.  Kinetic Studies of Polyhydroxybutyrate Granule Formation in Wautersia eutropha H16 by Transmission Electron Microscopy 
Journal of Bacteriology  2005;187(11):3814-3824.
Wautersia eutropha, formerly known as Ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (PHB) as insoluble granules inside the cell when nutrients other than carbon are limited. In this paper, we report findings from kinetic studies of granule formation and degradation in W. eutropha H16 obtained using transmission electron microscopy (TEM). In nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the first time. At the center of the cells, dark-stained “mediation elements” with small granules attached were observed. These mediation elements are proposed to serve as nucleation sites for granule initiation. TEM images also revealed that when W. eutropha cells were introduced into nitrogen-limited medium from nutrient-rich medium, the cell size increased two- to threefold, and the cells underwent additional volume changes during growth. Unbiased stereology was used to analyze the two-dimensional TEM images, from which the average volume of a W. eutropha H16 cell and the total surface area of granules per cell in nutrient-rich and PHB production media were obtained. These parameters were essential in the calculation of the concentration of proteins involved in PHB formation and utilization and their changes with time. The extent of protein coverage of the granule surface area is presented in the accompanying paper (J. Tian, A. He, A. Lawrence, P. Liu, N. Watson, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3825-3832, 2005).
PMCID: PMC1112049  PMID: 15901706
17.  pB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus 
BMC Microbiology  2004;4:15.
Gram-positive bacteria of the genus Rhodococcus have shown an extraordinary capacity for metabolizing recalcitrant organic compounds. One hindrance to the full exploitation of Rhodococcus is the dearth of genetic tools available for strain manipulation. To address this issue, we sought to develop a plasmid-based system for genetic manipulation of a variety of Rhodococcus strains.
We isolated and sequenced pB264, a 4,970 bp cryptic plasmid from Rhodococcus sp. B264-1 with features of a theta-type replication mechanism. pB264 was nearly identical to pKA22, a previously sequenced but uncharacterized cryptic plasmid. Derivatives of pB264 replicate in a diverse range of Rhodococcus species, showing that this plasmid does not bear the same host range restrictions that have been exhibited by other theta replicating plasmids. Replication or maintenance of pB264 is inhibited at 37°C, making pB264 useful as a suicide vector for genetic manipulation of Rhodococcus. A series of deletions revealed that ca. 1.3 kb from pB264 was sufficient to support replication and stable inheritance of the plasmid. This region includes two open reading frames that encode functions (RepAB) that can support replication of pB264 derivatives in trans. Rhodococcus sp. B264-1 will mobilize pB264 into other Rhodococcus species via conjugation, making it possible to genetically modify bacterial strains that are otherwise difficult to transform. The cis-acting element (oriT) required for conjugal transfer of pB264 resides within a ca. 0.7 kb region that is distinct from the regions responsible for replication.
Shuttle vectors derived from pB264 will be useful for genetic studies and strain improvement in Rhodococcus, and will also be useful for studying the processes of theta replication and conjugal transfer among actinomycetes.
PMCID: PMC419973  PMID: 15084226
18.  Ralstonia eutropha H16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes 
Journal of Bacteriology  2003;185(13):3788-3794.
Intracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.
PMCID: PMC161563  PMID: 12813072
19.  The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production 
Journal of Bacteriology  2002;184(1):59-66.
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.
PMCID: PMC134771  PMID: 11741844
20.  Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells 
Journal of Bacteriology  2001;183(14):4217-4226.
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.
PMCID: PMC95311  PMID: 11418562
21.  Development and Validation of Corynebacterium DNA Microarrays 
We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.
PMCID: PMC92872  PMID: 11319117
22.  New Insight into the Role of the PhaP Phasin of Ralstonia eutropha in Promoting Synthesis of Polyhydroxybutyrate 
Journal of Bacteriology  2001;183(7):2394-2397.
Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.
PMCID: PMC95152  PMID: 11244085
23.  Characterization of Lactobacillus bulgaricus Bacteriophage ch2 
Bacteriophage ch2, a virulent bacteriophage of Lactobacillus bulgaricus CH2, was characterized according to its morphology, genome size, structural proteins, and growth kinetics. Electron micrographs revealed that bacteriophage ch2 has an icosahedral head of 50-nm diameter and a long tail of 170 nm. Its genome is linear and 35 kilobases in length, and its structural proteins consist of two major and eight minor proteins. One-step growth kinetics of bacteriophage ch2 under optimal conditions (45°C in MRS medium [Oxoid Ltd.]) showed that the latent time was 40 min, the rise period was 15 min, and the burst size was 130 bacteriophages per cell. To monitor the effects of bacteriophage infection on host growth and β-galactosidase production, the absorbance of the culture and the β-galactosidase activity were followed during the infection cycle. Before lysis the infected culture continued to grow and produce β-galactosidase at the same rate as the uninfected culture.
PMCID: PMC202617  PMID: 16347627
24.  Ionizing Radiation Damage to the Folded Chromosome of Escherichia coli K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid 
Journal of Bacteriology  1979;138(2):486-491.
The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475–485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.
PMCID: PMC218202  PMID: 374389
25.  Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media1 
Journal of Bacteriology  1973;115(2):522-528.
Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.
PMCID: PMC246279  PMID: 4579871

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