Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Application of a Specific and Sensitive Radiometric Assay for Microbial Lipase Activities in Marine Water Samples from the Lagoon of Nouméa

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [³H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [³H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA·liter⁻¹·h⁻¹ and from 0.76 to 0.23 nmol of ³H-FFA·liter⁻¹·h⁻¹, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 μg of C·liter⁻¹·h⁻¹). However, the ratio of MUF-oleate activities to [³H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.     

Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths of Escherichia coli KO11 by Expression of Pseudomonas putida estZ Esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter⁻¹. Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. The K_m and V_max for α-naphthyl acetate were 18 μM and 48.1 μmol·min⁻¹·mg of protein⁻¹, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol·min⁻¹·mg of protein⁻¹), followed by ethyl acetate (66 μmol·min⁻¹·mg of protein⁻¹). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter⁻¹.     

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, ^7MeG^5′-ppp^5′-A_2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and ^7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC_n and ^7MeGpppAC_n (1≤n≤9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1≤n≤7) in quantities of up to 100nmol starting from 200nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.     

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T_m value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t_1/2 values of>24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t_1/2=1631min) compared with FRNA (t_1/2=53.2min) and MeRNA (t_1/2=187min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.     

Progress in the discovery of selective, high affinity A2B adenosine receptor antagonists as clinical candidates

The selective, high affinity A_2B adenosine receptor (AdoR) antagonists that were synthesized by several research groups should aid in determining the role of the A_2B AdoR in inflammatory diseases like asthma or rheumatoid arthritis (RA) and angiogenic diseases like diabetic retinopathy or cancer. CV Therapeutics scientists discovered the selective, high affinity A_2B AdoR antagonist 10, a 8-(4-pyrazolyl)-xanthine derivative [CVT-6883, K_i(hA_2B)=22 nM; K_i(hA₁)=1,940 nM; K_i(hA_2A)=3,280; and K_i(hA₃)=1,070 nM] that has favorable pharmacokinetic (PK) properties (t_1/2=4 h and F>35% rat). Compound 10 demonstrated functional antagonism at the A_2B AdoR (K_B=6 nM) and efficacy in a mouse model of asthma. In two phase 1 clinical trials, CVT-6883 was found to be safe, well tolerated, and suitable for once daily dosing. A second compound 20, 8-(5-pyrazolyl)-xanthine, has been nominated for development from Baraldi’s group in conjunction with King Pharmaceuticals that has favorable A_2B AdoR affinity and selectivity [K_i(hA_2B)=5.5 nM; K_i(hA₁) >1,000 nM; K_i(hA_2A) >1,000; and K_i(hA₃) >1,000 nM], and it has been demonstrated to be a functional antagonist. A third compound 32, a 2-aminopyrimidine, from the Almirall group has high A_2B AdoR affinity and selectivity [K_i(hA_2B)=17 nM; K_i(hA₁) >1,000 nM; K_i(hA_2A) >2,500; and K_i(hA₃) >1,000 nM], and 32 has been moved into preclinical safety testing. Since three highly selective, high affinity A_2B AdoR antagonists have been nominated for development with 10 (CVT-6883) being the furthest along in the development process, the role of the A_2B AdoR in various disease states will soon be established.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.     

Meta-Analysis of 28,141 Individuals Identifies Common Variants within Five New Loci That Influence Uric Acid Concentrations

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p=5.2×10⁻²⁰¹), ABCG2 (p=3.1×10⁻²⁶), SLC17A1 (p=3.0×10⁻¹⁴), SLC22A11 (p=6.7×10⁻¹⁴), SLC22A12 (p=2.0×10⁻⁹), SLC16A9 (p=1.1×10⁻⁸), GCKR (p=1.4×10⁻⁹), LRRC16A (p=8.5×10⁻⁹), and near PDZK1 (p=2.7×10⁻⁹). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p=4.0×10⁻²⁶) and propionyl-L-carnitine (p=5.0×10⁻⁸) concentrations, which in turn were associated with serum UA levels (p=1.4×10⁻⁵⁷ and p=8.1×10⁻⁵⁴, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.     

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, ¹⁴C-labeled acyl-HSLs were mineralized to ¹⁴CO₂ rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent K_m of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol·h⁻¹·g of fresh weight soil⁻¹. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO₂ but that the two processes were well coupled kinetically. A most probable number of 4.6 × 10⁵ cells·g of turf soil⁻¹ degraded physiologically relevant amounts of hexanoyl-[1-¹⁴C]HSL to ¹⁴CO₂. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.     

Specificity of the STAT4 genetic association for severe disease manifestations of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n=1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r²=0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF)=31.1% in SLE cases compared with 22.5% in controls (OR=1.56, p=10⁻¹⁶). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF=35.1%, OR=1.86, p<10⁻¹⁹), nephritis (MAF=34.3%, OR=1.80, p<10⁻¹¹), and age at diagnosis<30 years (MAF=33.8%, OR=1.77, p<10⁻¹³). An association with severe nephritis was even more striking (MAF=39.2%, OR=2.35, p<10⁻⁴ in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.     

Meta-Analysis of Genome-Wide Scans for Human Adult Stature Identifies Novel Loci and Associations with Measures of Skeletal Frame Size

Recent genome-wide (GW) scans have identified several independent loci affecting human stature, but their contribution through the different skeletal components of height is still poorly understood. We carried out a genome-wide scan in 12,611 participants, followed by replication in an additional 7,187 individuals, and identified 17 genomic regions with GW-significant association with height. Of these, two are entirely novel (rs11809207 in CATSPER4, combined P-value=6.1×10⁻⁸ and rs910316 in TMED10, P-value=1.4×10⁻⁷) and two had previously been described with weak statistical support (rs10472828 in NPR3, P-value=3×10⁻⁷ and rs849141 in JAZF1, P-value=3.2×10⁻¹¹). One locus (rs1182188 at GNA12) identifies the first height eQTL. We also assessed the contribution of height loci to the upper- (trunk) and lower-body (hip axis and femur) skeletal components of height. We find evidence for several loci associated with trunk length (including rs6570507 in GPR126, P-value=4×10⁻⁵ and rs6817306 in LCORL, P-value=4×10⁻⁴), hip axis length (including rs6830062 at LCORL, P-value=4.8×10⁻⁴ and rs4911494 at UQCC, P-value=1.9×10⁻⁴), and femur length (including rs710841 at PRKG2, P-value=2.4×10⁻⁵ and rs10946808 at HIST1H1D, P-value=6.4×10⁻⁶). Finally, we used conditional analyses to explore a possible differential contribution of the height loci to these different skeletal size measurements. In addition to validating four novel loci controlling adult stature, our study represents the first effort to assess the contribution of genetic loci to three skeletal components of height. Further statistical tests in larger numbers of individuals will be required to verify if the height loci affect height preferentially through these subcomponents of height.     

Admixture Mapping of 15,280 African Americans Identifies Obesity Susceptibility Loci on Chromosomes 5 and X

The prevalence of obesity (body mass index (BMI) ≥30 kg/m²) is higher in African Americans than in European Americans, even after adjustment for socioeconomic factors, suggesting that genetic factors may explain some of the difference. To identify genetic loci influencing BMI, we carried out a pooled analysis of genome-wide admixture mapping scans in 15,280 African Americans from 14 epidemiologic studies. Samples were genotyped at a median of 1,411 ancestry-informative markers. After adjusting for age, sex, and study, BMI was analyzed both as a dichotomized (top 20% versus bottom 20%) and a continuous trait. We found that a higher percentage of European ancestry was significantly correlated with lower BMI (ρ=−0.042, P=1.6×10⁻⁷). In the dichotomized analysis, we detected two loci on chromosome X as associated with increased African ancestry: the first at Xq25 (locus-specific LOD=5.94; genome-wide score=3.22; case-control Z=−3.94); and the second at Xq13.1 (locus-specific LOD=2.22; case-control Z=−4.62). Quantitative analysis identified a third locus at 5q13.3 where higher BMI was highly significantly associated with greater European ancestry (locus-specific LOD=6.27; genome-wide score=3.46). Further mapping studies with dense sets of markers will be necessary to identify the alleles in these regions of chromosomes X and 5 that may be associated with variation in BMI.     

Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C·t value (product of the disinfectant concentration and contact time) of 60 mg·min·liter⁻¹, frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C·t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4°C to 25°C) and a decrease in pH result in better inactivation.     

NRXN3 Is a Novel Locus for Waist Circumference: A Genome-Wide Association Study from the CHARGE Consortium

Central abdominal fat is a strong risk factor for diabetes and cardiovascular disease. To identify common variants influencing central abdominal fat, we conducted a two-stage genome-wide association analysis for waist circumference (WC). In total, three loci reached genome-wide significance. In stage 1, 31,373 individuals of Caucasian descent from eight cohort studies confirmed the role of FTO and MC4R and identified one novel locus associated with WC in the neurexin 3 gene [NRXN3 (rs10146997, p=6.4×10⁻⁷)]. The association with NRXN3 was confirmed in stage 2 by combining stage 1 results with those from 38,641 participants in the GIANT consortium (p=0.009 in GIANT only, p=5.3×10⁻⁸ for combined analysis, n=70,014). Mean WC increase per copy of the G allele was 0.0498 z-score units (0.65 cm). This SNP was also associated with body mass index (BMI) [p=7.4×10⁻⁶, 0.024 z-score units (0.10 kg/m²) per copy of the G allele] and the risk of obesity (odds ratio 1.13, 95% CI 1.07–1.19; p=3.2×10⁻⁵ per copy of the G allele). The NRXN3 gene has been previously implicated in addiction and reward behavior, lending further evidence that common forms of obesity may be a central nervous system-mediated disorder. Our findings establish that common variants in NRXN3 are associated with WC, BMI, and obesity.     

Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h⁻¹ at 100 g of glucose·liter⁻¹). In aerated batch cultures grown on 400 g of glucose·liter⁻¹, this engineered S. cerevisiae strain produced over 200 g of glycerol·liter⁻¹, corresponding to a molar yield of glycerol on glucose close to unity.     

Local and global effects of strong DNA bending induced during molecular dynamics simulations

DNA bending plays an important role in many biological processes, but its molecular and energetic details as a function of base sequence remain to be fully understood. Using a recently developed restraint, we have studied the controlled bending of four different B-DNA oligomers using molecular dynamics simulations. Umbrella sampling with the AMBER program and the recent parmbsc0 force field yield free energy curves for bending. Bending 15-base pair oligomers by 90° requires roughly 5kcalmol⁻¹, while reaching 150° requires of the order of 12kcalmol⁻¹. Moderate bending occurs mainly through coupled base pair step rolls. Strong bending generally leads to local kinks. The kinks we observe all involve two consecutive base pair steps, with disruption of the central base pair (termed Type II kinks in earlier work). A detailed analysis of each oligomer shows that the free energy of bending only varies quadratically with the bending angle for moderate bending. Beyond this point, in agreement with recent experiments, the variation becomes linear. An harmonic analysis of each base step yields force constants that not only vary with sequence, but also with the degree of bending. Both these observations suggest that DNA is mechanically more complex than simple elastic rod models would imply.     

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.     

Distributed control for recruitment, scanning and subunit joining steps of translation initiation

Protein synthesis utilizes a large proportion of the available free energy in the eukaryotic cell and must be precisely controlled, yet up to now there has been no systematic rate control analysis of the in vivo process. We now present a novel study of rate control by eukaryotic translation initiation factors (eIFs) using yeast strains in which chromosomal eIF genes have been placed under the control of the tetO7 promoter system. The results reveal that, contrary to previously published reports, control of the initiation pathway is distributed over all of the eIFs, whereby rate control (the magnitude of their respective component control coefficients) follows the order: eIF4G>eIF1A>eIF4E>eIF5B. The apparent rate control effects of eIFs observed in standard cell-free extract experiments, on the other hand, do not accurately reflect the steady state in vivo data. Overall, this work establishes the first quantitative control framework for the study of in vivo eukaryotic translation.     

Cooccurrence of ScDSP Gene Expression, Cell Death, and DNA Fragmentation in a Marine Diatom, Skeletonema costatum

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5′ and 3′ untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day⁻¹, the ScDSP mRNA level was at 2 μmol·mole 18S rRNA⁻¹. In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day⁻¹, ScDSP mRNA increased dramatically to 668 μmol·mole 18S rRNA⁻¹, and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.