Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is ¹³C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a ¹³C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.

As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps ². First, we grow cells with ¹³C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways^{3, 4}. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active ². Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.

¹³C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism¹. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Chemosphere

To advance the accuracy of bioremediation measurements, it is useful before specific experiments to attribute or estimate the influence of both experimental as well as field conditions on the expected magnitudes of microbial degradation rate coefficients. This paper analyzes the numerical contribution, or influence, of categories of conditions, such as bacterial adaptive state, electron acceptor type, mixing, generalized sorption conditions, and biodegradation temperature, on published phenanthrene biodegradation rates as an example of our regression approach. A fundamental microbial degradation rate equation is transformed to an additive model, then using multiple linear regression on published data, coefficients (of categorical variables) and a linear model are presented that estimate first-order biodegradation rate coefficients to within a factor of 3. Numerical estimates of how much bacterial adaptive state and presence of a sorption phase, the two most statistically significant factors, alter the phenanthrene biodegradation rate are presented. The influence of some measurement or field conditions, for example, the influence of oxygen reduction versus optimal nitrate reduction, cannot be distinguished statistically given the available data and range. The regression model is tested using conditions from newly published papers to estimate a priori the expected rate, which compares very favorably to measurements reported in the papers. Due to limited published data and range for extreme cases, the current coefficients do not apply to degradation of very aged phenanthrene nor very low concentrations of electron acceptors. As estimating tools, however, the coefficients themselves and the regression approach have very beneficial roles in design of experiments for both laboratory and field settings. Our method can be applied to other PAHs as sufficient data become available.

Environmental Science & Technology

Experimental measurements demonstrated that rates of in situ microbial anaerobic biodegradation of phenanthrene in undisturbed marine sediments were enhanced when controlled-release electron acceptors (i.e., nitrate and sulfate (for comparison)) were employed. The experimental method used whole and interval cores injected with radiolabeled 14C-phenanthrene, which were incubated, sacrificed, and processed for 14CO2 recovery to determine degradation rates. Nitrocellulose and CaSO4 were formulated to release the electron acceptors into the sediments at rates consistent with bacterial utilization (i.e., that avoided inhibition observed previously at high soluble nitrate (although not sulfate) concentrations). The controlled-release of both compounds, measured in collateral experiments, enhanced the natural anaerobic phenanthrene biodegradation rates by factors up to 2-3. Biodegradation via sulfate reduction was most rapid in the early stages (24 days) of experiments, consistent with reports that many marine bacteria in submerged sediments are sulfate reducers. In comparison, in longer experiments (after 42 days), the anaerobic biodegradation rates were observed at least as high with the addition of nitrocellulose (over 40% of added phenanthrene recovered as 14CO2). Both nitrate and sulfate reduction was observed during anaerobic incubation, although the presence of nitrate seemed to reduce the sulfate reduction. The studied forms of the controlled-release nitrate and sulfate may provide capping amendments to decontaminate marine harbor sediments.

Biotechnology and Bioengineering

The growth and Cr(VI) reduction by Shewanella oneidensis MR-1 was examined using a mini-bioreactor system that independently monitors and controls pH, dissolved oxygen (DO), and temperature for each of its 24, 10-mL reactors. Independent monitoring and control of each reactor in the cassette allows the exploration of a matrix of environmental conditions known to influence S. oneidensis chromium reduction. S. oneidensis MR-1 grew in minimal medium without amino acid or vitamin supplementation under aerobic conditions but required serine and glycine supplementation under anaerobic conditions. Growth was inhibited by DO concentrations >80%. Lactate transformation to acetate was enhanced by low concentration of DO during the logarithmic growth phase. Between 11 and 35 degrees C, the growth rate obeyed the Arrhenius reaction rate-temperature relationship, with a maximum growth rate occurring at 35 degrees C. S. oneidensis MR-1 was able to grow over a wide range of pH (6-9). At neutral pH and temperatures ranging from 30 to 35 degrees C, S. oneidensis MR-1 reduced 100 microM Cr(VI) to Cr(III) within 20 min in the exponential growth phase, and the growth rate was not affected by the addition of chromate; it reduced chromate even faster at temperatures between 35 and 39 degrees C. At low temperatures (<25 degrees C), acidic (pH < 6.5), or alkaline (pH > 8.5) conditions, 100 microM Cr(VI) strongly inhibited growth and chromate reduction. The mini-bioreactor system enabled the rapid determination of these parameters reproducibly and easily by performing very few experiments. Besides its use for examining parameters of interest to environmental remediation, the device will also allow one to quickly assess parameters for optimal production of recombinant proteins or secondary metabolites.

Marine Pollution Bulletin

A whole-core injection method was used to determine depth-related rates of microbial mineralization of (14)C-phenanthrene added to both contaminated and clean marine sediments of Puget Sound, WA. For 26-day incubations under micro-aerobic conditions, conversions of (14)C-phenanthrene to (14)CO(2) in heavily PAH-contaminated sediments from two sites in Eagle Harbor were much higher (up to 30%) than those in clean sediments from nearby Blakely Harbor (<3%). The averaged (14)C-phenanthrene degradation rates in the surface sediment horizons (0-3 cm) were more rapid (2-3 times) than in the deeper sediment horizons examined (>6 cm), especially in the most PAH polluted EH9 site. Differences in mineralization were associated with properties of the sediments as a function of sediment depth, including grain-size distribution, PAH concentration, total organic matter and total bacterial abundance. When strictly anaerobic incubations (in N(2)/H(2)/CO(2) atmosphere) were used, the phenanthrene biodegradation rates at all sediment depths were two times slower than under micro-aerobic conditions, with methanogenesis observed after 24 days. The main rate-limiting factor for phenanthrene degradation under anaerobic conditions appeared to be the availability of suitable electron acceptors. Addition of calcium sulfate enhanced the first order rate coefficient (k(1) increased from 0.003 to 0.006 day(-1)), whereas addition of soluble nitrate, even at very low concentration (<0.5 mM), inhibited mineralization. Long-term storage of heavily polluted Eagle Harbor sediment as intact cores under micro-aerobic conditions also appeared to enhance anaerobic biodegradation rates (k(1) up to 0.11 day(-1)).

Biotechnology and Bioengineering

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.

Applied and Environmental Microbiology

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

Journal of Bacteriology

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.

Nano Letters

The effects of four types of fullerene compounds (C60, C60-OH, C60-COOH, C60-NH2) were examined on two model microorganisms (Escherichia coli W3110 and Shewanella oneidensis MR-1). Positively charged C60-NH2 at concentrations as low as 10 mg/L inhibited growth and reduced substrate uptake for both microorganisms. Scanning electron microscopy (SEM) revealed damage to cellular structures. Neutrally charged C60 and C60-OH had mild negative effects on S. oneidensis MR-1, whereas the negatively charged C60-COOH did not affect either microorganism's growth. The effect of fullerene compounds on global metabolism was further investigated using [3-13C]L-lactate isotopic labeling, which tracks perturbations to metabolic reaction rates in bacteria by examining the change in the isotopic labeling pattern in the resulting metabolites (often amino acids).1-3 The 13C isotopomer analysis from all fullerene-exposed cultures revealed no significant differences in isotopomer distributions from unstressed cells. This result indicates that microbial central metabolism is robust to environmental stress inflicted by fullerene nanoparticles. In addition, although C60-NH2 compounds caused mechanical stress on the cell wall or membrane, both S. oneidensis MR-1 and E. coli W3110 can efficiently alleviate such stress by cell aggregation and precipitation of the toxic nanoparticles. The results presented here favor the hypothesis that fullerenes cause more membrane stress 4-6 than perturbation to energy metabolism.7.

Chemosphere

Rates of 14C-phenanthrene mineralization in contaminated, undisturbed marine sediments were measured using the whole core injection method to assess microbial natural attenuation activity as a function of sediment depth. Submerged sediments were sampled from Eagle Harbor, a marine superfund site in Puget Sound. Experiments show significant biodegradation activities (0.0012-0.0036 day(-1)) in the sediment horizons from 0 to 10 cm. The purpose and scope of this paper is to evaluate the range of experimental conditions giving valid results; a mathematical simulation described competing contaminant 14C-phenanthrene diffusion and simultaneous biodegradation (Monod kinetics), both retarded by sorption. The effect of aging was examined with two sorption models in presumed pseudo-homogenous sediments having effective properties. The simulation predictions provide quantitative guidelines for the successful use of the whole core injection method. (1) The effective Monod constant KS' in sediment is increased by a large partition coefficient KP between sediment and water and makes the apparent 14C-phenanthrene biodegradation approach first-order kinetics. (2) When KS'>1 mg(-1) l(-1), the measured 14C-phenanthrene biodegradation extent is biased by inadequately distributed injected tracer only when less than 7% of the sediment horizon is initially probed and mixed with injected tracer. (3) A short incubation time (<20 days) is necessary when a mobile indicator, e.g., gaseous 14CO2, is used. For longer incubation times, predictions show that a 14CO2 indicator diffuses to adjacent horizons, thus smearing the depth profile of biodegradation. (4) This method employing a radiolabeled tracer provides accurate biodegradation rates for freshly contaminated sediments, and represents an upper limit to the natural phenanthrene biodegradation extents if the contaminant is aged over 50 days.

Applied and Environmental Microbiology

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.

Analytical Chemistry

13C-Based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus, metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism Escherichia coli expressing a plasmid-borne, His-tagged green fluorescent protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

Current Opinion in Microbiology

Measurements of low molecular weight metabolites have been increasingly incorporated in the characterization of cellular physiology, qualitative studies in functional genomics, and stress response determination. The application of cutting edge analytical technologies to the measurement of metabolites and the changes in metabolite concentrations under defined conditions have helped illuminate the effects of perturbations in pathways of interest, such as the tricarboxylic acid cycle, as well as unbiased characterizations of microbial stress responses as a whole. Owing to the complexity of microbial metabolite extracts and the large number of metabolites therein, advanced and high-throughput separation techniques in gas chromatography, liquid chromatography, and capillary electrophoresis have been coupled to mass spectrometry - usually high-resolution mass spectrometry, but not exclusively - to make these measurements.

Biotechnology and Bioengineering

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)(-1) h(-1)) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64 +/- 3 to 25 +/- 2 and from 30 +/- 2 to 19 +/- 2, respectively. The carbon flux under micro-aerobic growth was directed to ethanol, L-lactate (> 99% optical purity), acetate, and formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38 +/- 0.07 mol mol(-1) glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

Mass Spectrometry Reviews

Metabolic flux analysis via (13)C labeling ((13)C MFA) quantitatively tracks metabolic pathway activity and determines overall enzymatic function in cells. Three core techniques are necessary for (13)C MFA: (1) a steady state cell culture in a defined medium with labeled-carbon substrates; (2) precise measurements of the labeling pattern of targeted metabolites; and (3) evaluation of the data sets obtained from mass spectrometry measurements with a computer model to calculate the metabolic fluxes. In this review, we summarize recent advances in the (13)C-flux analysis technologies, including mini-bioreactor usage for tracer experiments, isotopomer analysis of metabolites via high resolution mass spectrometry (such as GC-MS, LC-MS, or FT-ICR), high performance and large-scale isotopomer modeling programs for flux analysis, and the integration of fluxomics with other functional genomics studies. It will be shown that there is a significant value for (13)C-based metabolic flux analysis in many biological research fields.

Biotechnology and Bioengineering

Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and freshwater environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal medium with [3-13C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23-0.29 h(-1)) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo-steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636-640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate.

Biotechnology Letters

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O(2) transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD(+); (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways.

Applied and Environmental Microbiology

Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C(5) and C(6) sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via (13)C-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining (13)C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities.

Journal of Bacteriology

Members of the genus "Dehalococcoides" are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in "Dehalococcoides ethenogenes" strain 195 via (13)C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the (13)C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO(2) is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the (13)C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

Biotechnology Progress

An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time approximately 3 h), salt stressed minimal medium (doubling time approximately 6 h), and minimal medium with amino acid supplementation (doubling time approximately 1.5 h). (13)C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, 10 transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

PloS One

The Roseobacter clade of aerobic marine proteobacteria, which compose 10-25% of the total marine bacterial community, has been reported to fix CO(2), although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO(2) assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

Microbiology

Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile for leucine and isoleucine when Cyanothece 51142 was grown mixotrophically using 2-(13)C-labelled glycerol as the main carbon source. This indicated that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway was confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in the normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse-transcription PCR indicated that the cce_0248 gene is expressed and its transcriptional level is lower in medium with isoleucine than in isoleucine-free medium. Additionally, a blast search for citramalate synthase and threonine ammonia-lyase implies that this alternative isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic micro-organisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway.

Biotechnology Progress

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully (13)C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

The Science of the Total Environment

The toxicity of Cu-doped TiO(2) nanoparticles (NPs, 20nm), synthesized by a flame aerosol reactor, to Mycobacterium smegmatis and Shewanella oneidensis MR-1, is the primary focus of this study. Both doped and non-doped TiO(2) NPs (20nm) tended to agglomerate in the medium solution, and therefore did not penetrate into the cell and damage cellular structures. TiO(2) particles (<100mg/L) did not apparently interfere with the growth of the two species in aqueous cultures. Cu-doped TiO(2) NPs (20mg/L) significantly reduced the M. smegmatis growth rate by three fold, but did not affect S. oneidensis MR-1 growth. The toxicity of Cu-doped TiO(2) NPs was driven by the release of Cu(2+) from the parent NPs. Compared to equivalent amounts of Cu(2+), Cu-doped TiO(2) NPs exhibited higher levels of toxicity to M. smegmatis (P-value<0.1). Addition of EDTA in the culture appeared to significantly decrease the anti-mycobacterium activity of Cu-doped TiO(2) NPs. S. oneidensis MR-1 produced a large amount of extracellular polymeric substances (EPS) under NP stress, especially extracellular protein. Therefore, S. oneidensis MR-1 was able to tolerate a much higher concentration of Cu(2+) or Cu-doped TiO(2) NPs. S. oneidensis MR-1 also adsorbed NPs on cell surface and enzymatically reduced ionic copper in culture medium with a remediating rate of 61microg/(liter x OD(600) x hour) during its early exponential growth phase. Since the metal reducing Shewanella species can efficiently "clean" metal-oxide NPs, the activities of such environmentally relevant bacteria may be an important consideration for evaluating the ecological risk of metal-oxide NPs.

Environmental Science & Technology

The antimicrobial activity of ZnO nanoparticles (NPs) was investigated under aquatic and aerosol exposure modes. ZnO NPs in aquatic media aggregated to micrometer-sized particles and did not interact with microorganisms effectively. Hence, the inhibition of microbial growth by nano-ZnO NPs (e.g., Mycobacterium smegmatis and Cyanothece 51142) in aquatic media was mainly attributable to dissolved zinc species. Shewanella oneidensis MR-1 and Escherichia coli were able to produce large amounts of extracellular polymeric substances, and their growth was not inhibited by ZnO NPs in aquatic media, even at high concentrations (>40 mg/L). On the other hand, when ZnO NPs were electrosprayed onto an E. coli biofilm so that NPs could be directly deposited onto the cell surface, the aerosol exposure dramatically reduced cellular viability. For example, an electrospray of ZnO NPs (20 nm) reduced the total number of viable E.coli cells by 57% compared to the control case, in which we electrosprayed only the buffer solution. However, electrospraying large-sized ZnO particles (480 nm) or nonsoluble TiO(2) NPs (20 nm) caused much less lethality to E. coli cells. The above observation implies that the aerosol method of exposing ZnO NPs to biological systems appears to have a much higher antimicrobial activity, and thus may lead to practical applications of employing a novel antimicrobial agent for airborne disease control.

Microbiology

The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light-dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via (13)C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 mumol photons m(-2) s(-1)) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenase-dependent hydrogen production [up to 25 mumol H(2) (mg chlorophyll)( -1) h(-1)], but strongly reduced phototrophic CO(2) utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO(2) via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis.

Chemosphere

This study used the electrospray method to create airborne droplets of metal oxide nanoparticles (NPs) and examined their anti-microbial activities, employing Escherichia coli as a model microbial species. We tested the anti-microbial activities of six metal oxide NPs (NiO, ZnO, Fe(2)O(3), Co(3)O(4), CuO, and TiO(2)) in both an aqueous culture medium and an aerosol exposure mode (spraying the particles directly onto the cell surface). In the aqueous medium, the both NPs and stressed E. coli cells severely aggregated. Only NiO NPs (>20 mgL(-1)) showed significant growth inhibition of E. coli ( approximately 30%). In contrast to aqueous exposure, where the direct interactions between NPs and bacteria were limited, aerosol exposure of three metal oxide NPs to E. coli enhanced NP toxicity to cells and dramatically reduced cellular viability. Electrospraying NiO, CuO, or ZnO NPs (20 nm, 20 microg, in 10 min) reduced the total number of living E. coli by more than 88%, 77% and 71%, respectively (compared to the control experiments). However, TiO(2), Co(3)O(4), and Fe(2)O(3) NPs showed no significant antibacterial activities in either the aqueous exposure mode or the aerosol exposure mode. The above observations suggest the potential application of electrosprayed metal oxide NPs to disinfect airborne pathogens.

The Journal of Biological Chemistry

The recently discovered heliobacteria are the only Gram-positive photosynthetic bacteria that have been cultured. One of the unique features of heliobacteria is that they have properties of both the photosynthetic green sulfur bacteria (containing the type I reaction center) and Clostridia (forming heat-resistant endospores). Most of the previous studies of heliobacteria, which are strict anaerobes and have the simplest known photosynthetic apparatus, have focused on energy and electron transfer processes. It has been assumed that like green sulfur bacteria, the major carbon flow in heliobacteria is through the (incomplete) reductive (reverse) tricarboxylic acid cycle, whereas the lack of CO(2)-enhanced growth has not been understood. Here, we report studies to fill the knowledge gap of heliobacterial carbon metabolism. We confirm that the CO(2)-anaplerotic pathway is active during phototrophic growth and that isoleucine is mainly synthesized from the citramalate pathway. Furthermore, to our surprise, our results suggest that the oxidative (forward) TCA cycle is operative and more active than the previously reported reductive (reverse) tricarboxylic acid cycle. Both isotopomer analysis and activity assays suggest that citrate is produced by a putative (Re)-citrate synthase and then enters the oxidative (forward) TCA cycle. Moreover, in contrast to (Si)-citrate synthase, (Re)-citrate synthase produces a different isomer of 2-fluorocitrate that is not expected to inhibit the activity of aconitase.

The Journal of Biological Chemistry

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80∼100 units for mixotrophic cultures grown on acetate and 200∼230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.

Environmental Science & Technology

To study potential ecological impacts of CO(2) leakage to shallow groundwater and soil/sediments from geologic CO(2) sequestration (GCS) sites, this work investigated the viability and metal reduction of Shewanella oneidensis MR-1 under CO(2) stress. While MR-1 could grow under high-pressure nitrogen gas (500 psi), the mix of 1% CO(2) with N(2) at total pressures of 15 or 150 psi significantly suppressed the growth of MR-1, compared to the N(2) control. When CO(2) partial pressures were over 15 psi, the growth of MR-1 stopped. The reduced bacterial viability was consistent with the pH decrease and cellular membrane damage under high pressure CO(2). After exposure to 150 psi CO(2) for 5 h, no viable cells survived, the cellular contents were released, and microscopy images confirmed significant cell structure deformation. However, after a relatively short exposure (25 min) to 150 psi CO(2), MR-1 could fully recover their growth within 24 h after the stress was removed, and the reduction of MnO(2) by MR-1 was observed right after the stress was removed. Furthermore, MR-1 survived better if the cells were aggregated rather than suspended, or if pH buffering minerals, such as calcite, were present. To predict the cell viability under different CO(2) pressures and exposure times, a two-parameter mathematical model was developed.

Biotechnology and Bioengineering

In the production of chemicals via microbial fermentation, achieving a high yield is one of the most important objectives. We developed a statistical model to analyze influential factors that determine product yield by compiling data obtained from engineered Escherichia coli developed within last 10 years. Using both numerical and ordinal variables (e.g., enzymatic steps, cultivation conditions, and genetic modifications) as input parameters, our model revealed that cultivation modes, nutrient supplementation, and oxygen conditions were the three significant factors for improving product yield. Generally, the model showed that product yield decreases as the number of enzymatic steps in the biosynthesis pathway increases (7-9% loss of yield per enzymatic step). Moreover, overexpression of enzymes or removal of competitive pathways (e.g., knockout) does not necessarily result in an amplification of product yield (P-value >0.1), possibly because of limited capacity in the biosynthesis pathway to accommodate an increase in flux. The model not only provides general guidelines for metabolic engineering and fermentation processes, but also allows a priori estimation and comparison of product yields under designed cultivation conditions. Biotechnol. Bioeng. © 2010 Wiley Periodicals, Inc.

Journal of Microbiological Methods

In this study, we developed a technique for delivering genes to microorganisms via electrospray of gold nanoparticles. During the electrospray process, charged monodisperse nano-droplets (a mixture of pET30a-GFP plasmid and nano-sized gold particles) were accelerated and deposited on a thin layer of non-competent Escherichia coli cells. Via antibiotic selection, transformed cells containing green fluorescent protein appeared on the agar plates. PCR amplification and restriction enzyme analysis further confirmed that pET30a-GFP plasmid had successfully been delivered into the non-competent E. coli cells. The transformation efficiencies were optimized under different electrospray conditions. Among several electrospray buffer solutions, CaCl(2) (0.01M) was found to be the best for gene delivery. Furthermore, gold nanoparticles (NPs, 50 nm diameter) significantly improved plasmid transformation efficiency by 5-7 fold (up to 2×10(6) CFU/μg plasmid) compared with that obtained using naked plasmid. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmids remained unchanged during the electrospray process, but cellular membrane integrity was reduced after being electrosprayed with gold NPs and CaCl(2) buffer solutions. This gene delivery method has the potential to work for many other microorganisms.

Journal of Biomedicine & Biotechnology

Metabolic flux analysis is a vital tool used to determine the ultimate output of cellular metabolism and thus detect biotechnologically relevant bottlenecks in productivity. ¹³C-based metabolic flux analysis (¹³C-MFA) and flux balance analysis (FBA) have many potential applications in biotechnology. However, noteworthy hurdles in fluxomics study are still present. First, several technical difficulties in both ¹³C-MFA and FBA severely limit the scope of fluxomics findings and the applicability of obtained metabolic information. Second, the complexity of metabolic regulation poses a great challenge for precise prediction and analysis of metabolic networks, as there are gaps between fluxomics results and other omics studies. Third, despite identified metabolic bottlenecks or sources of host stress from product synthesis, it remains difficult to overcome inherent metabolic robustness or to efficiently import and express nonnative pathways. Fourth, product yields often decrease as the number of enzymatic steps increases. Such decrease in yield may not be caused by rate-limiting enzymes, but rather is accumulated through each enzymatic reaction. Fifth, a high-throughput fluxomics tool hasnot been developed for characterizing nonmodel microorganisms and maximizing their application in industrial biotechnology. Refining fluxomics tools and understanding these obstacles will improve our ability to engineer highly efficient metabolic pathways in microbial hosts.

Biotechnology and Bioengineering

In the production of chemicals via microbial fermentation, achieving a high yield is one of the most important objectives. We developed a statistical model to analyze influential factors that determine product yield by compiling data obtained from engineered Escherichia coli developed within last 10 years. Using both numerical and ordinal variables (e.g., enzymatic steps, cultivation conditions, and genetic modifications) as input parameters, our model revealed that cultivation modes, nutrient supplementation, and oxygen conditions were the three significant factors for improving product yield. Generally, the model showed that product yield decreases as the number of enzymatic steps in the biosynthesis pathway increases (7-9% loss of yield per enzymatic step). Moreover, overexpression of enzymes or removal of competitive pathways (e.g., knockout) does not necessarily result in an amplification of product yield (P-value>0.1), possibly because of limited capacity in the biosynthesis pathway to accommodate an increase in flux. The model not only provides general guidelines for metabolic engineering and fermentation processes, but also allows a priori estimation and comparison of product yields under designed cultivation conditions.

Microbial Cell Factories

The robustness of Saccharomyces cerevisiae in facilitating industrial-scale production of ethanol extends its utilization as a platform to synthesize other metabolites. Metabolic engineering strategies, typically via pathway overexpression and deletion, continue to play a key role for optimizing the conversion efficiency of substrates into the desired products. However, chemical production titer or yield remains difficult to predict based on reaction stoichiometry and mass balance. We sampled a large space of data of chemical production from S. cerevisiae, and developed a statistics-based model to calculate production yield using input variables that represent the number of enzymatic steps in the key biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability.

Analytical Biochemistry

In a (13)C experiment for metabolic flux analysis ((13)C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ(13)C) were in the range of -35 to -92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of (13)C MFA.

The Science of the Total Environment

It has been shown that photocatalytic TiO(2) nanoparticles (NPs) can be used as an efficient anti-microbial agent under UV light due to generation of reactive oxygen species (ROS), while Shewanella oneidensis MR-1 is a metal-reducing bacterium highly susceptible to UV radiation. Interestingly, we found that the presence of Cu-doped TiO(2) NPs in the cultural medium dramatically increased the survival rates (based on colony-forming unit) of strain MR-1 by over 10,000-fold (incubation without shaking) and ~200 fold (incubation with shaking) after a 2-h exposure to UV light. Gene expression results (via qPCR measurement) indicated that the DNA repair gene recA in MR-1 was significantly induced by UV exposure (indicating cellular damage under UV stress), but the influence of NPs on recA expression was not statistically evident. Plausible explanations to NP attenuation of UV stresses are: 1. TiO(2) based NPs are capable of scattering and absorbing UV light and thus create a shading effect to protect MR-1 from UV radiation; 2. more importantly, Cu-doped TiO(2) NPs can co-agglomerate with MR-1 to form large flocs that improves cells' survival against the environmental stresses. This study improves our understanding of NP ecological impacts under natural solar radiation and provides useful insights to application of photocatalytic-NPs for bacterial disinfection.

Frontiers in Microbiology

Photosynthesis is the biological process that converts solar energy to biomass, bio-products, and biofuel. It is the only major natural solar energy storage mechanism on Earth. To satisfy the increased demand for sustainable energy sources and identify the mechanism of photosynthetic carbon assimilation, which is one of the bottlenecks in photosynthesis, it is essential to understand the process of solar energy storage and associated carbon metabolism in photosynthetic organisms. Researchers have employed physiological studies, microbiological chemistry, enzyme assays, genome sequencing, transcriptomics, and (13)C-based metabolomics/fluxomics to investigate central carbon metabolism and enzymes that operate in phototrophs. In this report, we review diverse CO(2) assimilation pathways, acetate assimilation, carbohydrate catabolism, the tricarboxylic acid cycle and some key, and/or unconventional enzymes in central carbon metabolism of phototrophic microorganisms. We also discuss the reducing equivalent flow during photoautotrophic and photoheterotrophic growth, evolutionary links in the central carbon metabolic network, and correlations between photosynthetic and non-photosynthetic organisms. Considering the metabolic versatility in these fascinating and diverse photosynthetic bacteria, many essential questions in their central carbon metabolism still remain to be addressed.

Applied and Environmental Microbiology

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.

Nanotechnology

The properties of Cu-doped TiO(2) nanoparticles (NPs) were independently controlled in a flame aerosol reactor by varying the molar feed ratios of the precursors, and by optimizing temperature and time history in the flame. The effect of the physico-chemical properties (dopant concentration, crystal phase and particle size) of Cu-doped TiO(2) nanoparticles on inactivation of Mycobacterium smegmatis (a model pathogenic bacterium) was investigated under three light conditions (complete dark, fluorescent light and UV light). The survival rate of M. smegmatis (in a minimal salt medium for 2 h) exposed to the NPs varied depending on the light irradiation conditions as well as the dopant concentrations. In dark conditions, pristine TiO(2) showed insignificant microbial inactivation, but inactivation increased with increasing dopant concentration. Under fluorescent light illumination, no significant effect was observed for TiO(2). However, when TiO(2) was doped with copper, inactivation increased with dopant concentration, reaching more than 90% (>3 wt% dopant). Enhanced microbial inactivation by TiO(2) NPs was observed only under UV light. When TiO(2) NPs were doped with copper, their inactivation potential was promoted and the UV-resistant cells were reduced by over 99%. In addition, the microbial inactivation potential of NPs was also crystal-phase-and size-dependent under all three light conditions. A lower ratio of anatase phase and smaller sizes of Cu-doped TiO(2) NPs resulted in decreased bacterial survival. The increased inactivation potential of doped TiO(2) NPs is possibly due to both enhanced photocatalytic reactions and leached copper ions.

Applied and Environmental Microbiology

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.

PLoS Computational Biology

Shewanella oneidensis MR-1 sequentially utilizes lactate and its waste products (pyruvate and acetate) during batch culture. To decipher MR-1 metabolism, we integrated genome-scale flux balance analysis (FBA) into a multiple-substrate Monod model to perform the dynamic flux balance analysis (dFBA). The dFBA employed a static optimization approach (SOA) by dividing the batch time into small intervals (i.e., ∼400 mini-FBAs), then the Monod model provided time-dependent inflow/outflow fluxes to constrain the mini-FBAs to profile the pseudo-steady-state fluxes in each time interval. The mini-FBAs used a dual-objective function (a weighted combination of "maximizing growth rate" and "minimizing overall flux") to capture trade-offs between optimal growth and minimal enzyme usage. By fitting the experimental data, a bi-level optimization of dFBA revealed that the optimal weight in the dual-objective function was time-dependent: the objective function was constant in the early growth stage, while the functional weight of minimal enzyme usage increased significantly when lactate became scarce. The dFBA profiled biologically meaningful dynamic MR-1 metabolisms: 1. the oxidative TCA cycle fluxes increased initially and then decreased in the late growth stage; 2. fluxes in the pentose phosphate pathway and gluconeogenesis were stable in the exponential growth period; and 3. the glyoxylate shunt was up-regulated when acetate became the main carbon source for MR-1 growth.