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1.  Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress 
Applied and Environmental Microbiology  2014;80(14):4226-4233.
Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
PMCID: PMC4068688  PMID: 24795373
2.  Carbon Flux Analysis by 13C Nuclear Magnetic Resonance To Determine the Effect of CO2 on Anaerobic Succinate Production by Corynebacterium glutamicum 
Applied and Environmental Microbiology  2014;80(10):3015-3024.
Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using 13C-labeled glucose and 13C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H+:organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.
PMCID: PMC4018904  PMID: 24610842
3.  Production and crystallization of α-phosphoglucomutase from Lactococcus lactis  
α-Phosphoglucomutase from L. lactis, a homologue of human phosphomannomutase 1, was produced and crystallized. X-ray diffraction data were collected to 1.5 Å resolution.
α-Phosphoglucomutase (α-PGM) is an enzyme that is essential for the growth of Lactococcus lactis. The enzyme links bacterial anabolism with sugar utilization through glycolysis by catalyzing the reversible interconversion of glucose 6-­phosphate and α-glucose 1-phosphate. The gene encoding α-PGM was cloned and overexpressed in L. lactis. The purified protein was functionally active and was crystallized with ammonium sulfate as a precipitant using vapour-diffusion and seeding techniques. Optimized crystals diffracted to 1.5 Å resolution at a synchrotron source.
PMCID: PMC3433211  PMID: 22949208
α-phosphoglucomutase; Lactococcus lactis
4.  “Cancer – Educate to Prevent” – High-School Teachers, the New Promoters of Cancer Prevention Education Campaigns 
PLoS ONE  2014;9(5):e96672.
Cancer is one of the leading causes of death worldwide, and thus represents a priority for national public health programs. Prevention has been assumed as the best strategy to reduce cancer burden, however most cancer prevention programs are implemented by healthcare professionals, which constrain range and educational impacts.
We developed an innovative approach for cancer prevention education focused on high-school biology teachers, considered privileged mediators in the socialization processes. A training program, “Cancer, Educate to Prevent” was applied, so that the teachers were able to independently develop and implement prevention campaigns focused on students and school-related communities. The program encompassed different educational modules, ranging from cancer biology to prevention campaigns design. Fifty-four teachers were empowered to develop and implement their own cancer prevention campaigns in a population up to five thousands students.
The success of the training program was assessed through quantitative evaluation – questionnaires focused on teachers' cancer knowledge and perceptions, before the intervention (pre-test) and immediately after (post-test). The projects developed and implemented by teachers were also evaluated regarding the intervention design, educational contents and impact on the students' knowledge about cancer. This study presents and discusses the results concerning the training program “Cancer, Educate to Prevent” and clearly shows a significant increase in teacher's cancer literacy (knowledge and perceptions) and teachers' acquired proficiency to develop and deliver cancer prevention campaigns with direct impact on students' knowledge about cancer.
This pilot study reinforces the potential of high-school teachers and schools as cancer prevention promoters and opens a new perspective for the development and validation of cancer prevention education strategies, based upon focused interventions in restricted targets (students) through non-health professionals (teachers).
PMCID: PMC4016009  PMID: 24817168
5.  Blastocystis sp. in splenic cysts: causative agent or accidental association? A unique case report 
Parasites & Vectors  2014;7:207.
Blastocystis sp. is one of the most prevalent parasites found in human stool and has been recently considered an opportunistic emerging pathogen in immunocompromised individuals. However, cases of invasive intestinal infections and skin rashes have been attributed to infection by Blastocystis sp in immunocompetent individuals, suggesting that it is an emerging parasite with pathogenic potential.
We present a case of a 22 year old female patient who complained of pain in the left hypochondrium. Ultrasonography and abdominal computed tomography scans showed two splenic cysts. The cyst fluid analysis demonstrated numerous Blastocystis sp.; PCR and DNA sequencing analyses confirmed the presence of Blastocystis subtype 3.
This is, to our knowledge, the first case report of the presence of Blastocystis subtype 3 in extra-intestinal organs and is strong evidence that Blastocystis sp. is potentially pathogenic and invasive. However, further studies are required to determine the pathogenicity of the parasite.
PMCID: PMC4026114  PMID: 24779380
Blastocystis spp; Immunocompetent individual; Pathogenicity; Splenic cyst
6.  Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus 
Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
PMCID: PMC3995056  PMID: 24782854
Halobacillus halophilus; glutamine synthetase; compatible solutes; osmoregulation; halophile
7.  Phenotypic divergence in reproductive traits of a moth population experiencing a phenological shift 
Ecology and Evolution  2013;3(15):5098-5108.
Allochrony that is reproductive isolation by time may further lead to divergence of reproductive adaptive traits in response to different environmental pressures over time. A unique “summer” population of the pine processionary moth Thaumetopoea pityocampa, reproductively isolated from the typical winter populations by allochronic differentiation, is here analyzed. This allochronically shifted population reproduces in the spring and develops in the summer, whereas “winter” populations reproduce in the late summer and have winter larval development. Both summer and winter populations coexist in the same pine stands, yet they face different climatic pressures as their active stages are present in different seasons. The occurrence of significant differences between the reproductive traits of the summer population and the typical winter populations (either sympatric or allopatric) is thus hypothesized. Female fecundity, egg size, egg covering, and egg parasitism were analyzed showing that the egg load was lower and that egg size was higher in the summer population than in all the studied winter populations. The scales that cover the egg batches of T. pityocampa differed significantly between populations in shape and color, resulting in a looser and darker covering in the summer population. The single specialist egg parasitoid species of this moth was almost missing in the summer population, and the overall parasitism rates were lower than in the winter population. Results suggest the occurrence of phenotypic differentiation between the summer population and the typical T. pityocampa winter populations for the life-history traits studied. This work provides an insight into how ecological divergence may follow the process of allochronic reproductive isolation.
PMCID: PMC3892371  PMID: 24455139
Egg parasitoids; egg size; fecundity; phenotypic divergence; scale covering; Thaumetopoea pityocampa.
8.  Comparative effectiveness and predictors of response to tumour necrosis factor inhibitor therapies in rheumatoid arthritis 
Rheumatology (Oxford, England)  2012;51(11):2020-2026.
Objectives. Adalimumab, etanercept and infliximab are effective TNF inhibitors (TNFis) in the treatment of RA, but no randomized clinical trials have compared the three agents. Prior observational data are not consistent. We compared their effectiveness over 1 year in a prospective cohort.
Methods. Analyses were performed on subjects’ first episode of TNFi use in the Rheumatic Diseases Portuguese Register, The primary outcome was the proportion of patients with European League Against Rheumatism good response sustained at two consecutive observations separated by 3 months during the first year of TNFi use. Comparisons were performed using conventional adjusted logistic regression, as well as matching subjects across the three agents using a propensity score. In addition, baseline predictors of treatment response to TNFi were identified.
Results. The study cohort included 617 RA patients, 250 starting etanercept, 206 infliximab and 161 adalimumab. Good response was achieved by 59.6% for adalimumab, 59.2% for etanercept and 51.9% for infliximab (P = 0.21). The modelled probability of good response did not significantly differ across agents (etanercept vs adalimumab OR = 0.97, 95% CI 0.55, 1.71; etanercept vs infliximab OR = 1.25, 95% CI 0.74, 2.12; infliximab vs adalimumab OR = 0.80, 95% CI 0.47, 1.36). Matched propensity score analyses also showed no significant treatment response differences. Greater educational attainment was a predictor of better response, while smoking, presence of ACPA, glucocorticoid use and worse physician assessment of disease activity at baseline each predicted a reduced likelihood of treatment response.
Conclusion. Over 1 year, we found no difference in effectiveness between adalimumab, etanercept and infliximab.
PMCID: PMC3475979  PMID: 22843791
RA; TNF inhibitors; comparative effectiveness; register; response predictors
9.  Production and crystallization of α-phosphoglucomutase from Lactococcus lactis. Corrigendum 
A correction is made to the article by Nogly et al. (2012). Acta Cryst. F68, 1113–1115.
Details of the beamline, detector and overall Wilson B in the article by Nogly et al. (2012, Acta Cryst. F68, 1113–1115) are corrected.
PMCID: PMC3818064
α-phosphoglucomutase; Lactococcus lactis; corrigendum
10.  Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency: regional experience and high incidence of carnitine deficiency 
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited defect in the mitochondrial fatty acid oxidation pathway, resulting in significant morbidity and mortality in undiagnosed patients.
Newborn screening (NBS) has considerably improved MCADD outcome, but the risk of complication remains in some patients. The aim of this study was to evaluate the relationship between genotype, biochemical parameters and clinical data at diagnosis and during follow-up, in order to optimize monitoring of these patients.
We carried out a multicenter study in southwest Europe, of MCADD patients detected by NBS. Evaluated NBS data included free carnitine (C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10 ratios, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises.
C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the prevalent c.985A > G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median value of C0 was significantly lower (23 μmol/L vs 36 μmol/L).
The average follow-up period was 43 months. To keep carnitine levels within the normal range, carnitine supplementation was required in 82% of patients, and for a longer period in patients homozygotes for the c.985A>G mutation than in patients with other genotypes (average 31 vs 18 months). Even with treatment, median C0 levels remained lower in homozygous patients than in those with other genotypes (14 μmol/L vs 22 μmol/L).
Two patients died and another three suffered a metabolic crisis, all of whom were homozygous for the c.985 A>G mutation.
Our data show a direct association between homozygosity for c.985A>G and lower carnitine values at diagnosis, and a higher dose of carnitine supplementation for maintenance within the normal range. This study contributes to a better understanding of the relationship between genotype and phenotype in newborn patients with MCADD detected through screening which could be useful in improving follow-up strategies and clinical outcome.
PMCID: PMC3718718  PMID: 23842438
L-carnitine; Metabolic decompensation; Mitochondrial fatty acid oxidation; Mutations; Newborn screening; Rare disease
11.  Metabolic and Transcriptional Analysis of Acid Stress in Lactococcus lactis, with a Focus on the Kinetics of Lactic Acid Pools 
PLoS ONE  2013;8(7):e68470.
The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H+-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by 13C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.
PMCID: PMC3700934  PMID: 23844205
12.  Structure and Non-Structure of Centrosomal Proteins 
PLoS ONE  2013;8(5):e62633.
Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at
PMCID: PMC3650010  PMID: 23671615
13.  Trichorhinophalangeal Syndrome Type I: A Patient with Two Novel and Different Mutations in the TRPS1 Gene 
Case Reports in Genetics  2013;2013:748057.
Background. Trichorhinophalangeal syndrome (TRPS) is an autosomal dominant skeletal dysplasia caused by defects involving the TRPS1 gene. Three types (TRPSs I, II, and III) have been described, exhibiting the common triad of hair, craniofacial, and skeletal abnormalities. TRPS II includes the additional characteristics of mental retardation and multiple exostoses. Case Report. We describe a sporadic case of TRPS type I in a child with two novel nonsense pathogenic mutations in the TRPS1 gene, both in heterozygosity—c.1198C>T (p. Gln400X) and c.2086C>T (p.Arg696X). None of these mutations were found in her parents. Clinical presentation included typical hair and facial features, as well as slight skeletal abnormalities. Discussion. There is a wide variability in clinical expression of TRPS I. Manifestations of the disease can be subtle, yet skeletal anomalies imply that TRPS I is more than an esthetic problem. Clinical and genetic diagnosis allows adequate followup and timely therapeutic procedures. When a single mutation was sufficient for the onset of the disease, our patient presented two different ones.
PMCID: PMC3652099  PMID: 23691375
14.  LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools 
Parasites & Vectors  2013;6:69.
Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species.
PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools.
Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli.
These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.
PMCID: PMC3622617  PMID: 23497666
Entamoeba; 18SrRNA; PCR; Molecular diagnostics; Multiplex assay
15.  Crystallization and preliminary X-ray analysis of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27 
The cloning, expression, purification, crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate phosphatase (MpgP) from T. thermophilus HB27 are reported. The stability of MpgP in solution was studied by size-exclusion chromatography and differential scanning fluorimetry assays.
Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallo­graphic studies. The crystals obtained belonged to the monoclinic space group P21, with unit-cell parameters a = 39.52, b = 70.68, c = 95.42 Å, β = 92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.
PMCID: PMC3053170  PMID: 21393850
mannosyl-3-phosphoglycerate phosphatase; Thermus thermophilus HB27; mannosylglycerate synthesis
16.  The Fate of Acetic Acid during Glucose Co-Metabolism by the Spoilage Yeast Zygosaccharomyces bailii 
PLoS ONE  2012;7(12):e52402.
Zygosaccharomyces bailii is one of the most widely represented spoilage yeast species, being able to metabolise acetic acid in the presence of glucose. To clarify whether simultaneous utilisation of the two substrates affects growth efficiency, we examined growth in single- and mixed-substrate cultures with glucose and acetic acid. Our findings indicate that the biomass yield in the first phase of growth is the result of the weighted sum of the respective biomass yields on single-substrate medium, supporting the conclusion that biomass yield on each substrate is not affected by the presence of the other at pH 3.0 and 5.0, at least for the substrate concentrations examined. In vivo 13C-NMR spectroscopy studies showed that the gluconeogenic pathway is not operational and that [2−13C]acetate is metabolised via the Krebs cycle leading to the production of glutamate labelled on C2, C3 and C4. The incorporation of [U-14C]acetate in the cellular constituents resulted mainly in the labelling of the protein and lipid pools 51.5% and 31.5%, respectively. Overall, our data establish that glucose is metabolised primarily through the glycolytic pathway, and acetic acid is used as an additional source of acetyl-CoA both for lipid synthesis and the Krebs cycle. This study provides useful clues for the design of new strategies aimed at overcoming yeast spoilage in acidic, sugar-containing food environments. Moreover, the elucidation of the molecular basis underlying the resistance phenotype of Z. bailii to acetic acid will have a potential impact on the improvement of the performance of S. cerevisiae industrial strains often exposed to acetic acid stress conditions, such as in wine and bioethanol production.
PMCID: PMC3532111  PMID: 23285028
18.  Production, crystallization and preliminary X-ray analysis of CTP:inositol-1-phosphate cytidylyltransferase from Archaeoglobus fulgidus  
The expression, purification, crystallization and preliminary X-ray diffraction analysis of CTP:inositol-1-phosphate cytidylyltransferase from A. fulgidus is described.
Archaeoglobus fulgidus, a hyperthermophilic archaeon, accumulates di-myo-inositol phosphate (DIP) in response to heat stress. Recently, the pathway for biosynthesis of DIP has been elucidated in this organism and involves a bifunctional enzyme that contains two domains: CTP:inositol-1-phosphate cytidylyltransferase (IPCT) as a soluble domain and di-myo-inositol-1,3′-phosphate-1-phosphate synthase (DIPPS) as a membrane domain. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of the IPCT domain from A. fulgidus in the apo form are reported. The crystals diffracted to 2.4 Å resolution using a synchrotron source and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 154.7, b = 83.9, c = 127.7 Å.
PMCID: PMC3001648  PMID: 21045295
CTP:inositol-1-phosphate cytidylyltransferase; Archaeoglobus fulgidus; compatible solutes; CDP-inositol; di-myo-inositol phosphate
19.  Morphological and Molecular Diagnosis of Anisakid Nematode Larvae from Cutlassfish (Trichiurus lepturus) off the Coast of Rio de Janeiro, Brazil 
PLoS ONE  2012;7(7):e40447.
Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank.
PMCID: PMC3392247  PMID: 22792329
20.  High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †  
Applied and Environmental Microbiology  2011;77(19):6826-6835.
Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.
PMCID: PMC3187077  PMID: 21841021
21.  Engineering Trehalose Synthesis in Lactococcus lactis for Improved Stress Tolerance ▿ †  
Applied and Environmental Microbiology  2011;77(12):4189-4199.
Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.
PMCID: PMC3131666  PMID: 21515730
22.  Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in (Hyper)Thermophiles▿† 
Journal of Bacteriology  2011;193(9):2177-2185.
Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent Km values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the Vmax determined at 90°C was 62.9 μmol · min−1 · mg of protein−1. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X4-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.
PMCID: PMC3133074  PMID: 21378188
23.  Crystallization and preliminary X-ray analysis of mannosyl-3-phosphoglycerate synthase from Thermus thermophilus HB27 
Mannosyl-3-phosphoglycerate synthase (MpgS) is a key enzyme in the biosynthesis of MG. Here, the purification, crystallization and preliminary crystallographic characterization of apo MpgS from Thermus thermophilus HB27 are reported.
Mannosylglycerate (MG) is a compatible solute that is widespread in marine organisms that are adapted to hot environments, with its intracellular pool generally increasing in response to osmotic stress. These observations suggest that MG plays a relevant role in osmoadaptation and thermoadaptation. The pathways for the synthesis of MG have been characterized in a number of thermophilic and hyperthermophilic organisms. Mannosyl-3-phosphoglycerate synthase (MpgS) is a key enzyme in the biosynthesis of MG. Here, the purification, crystallization and preliminary crystallographic characterization of apo MpgS from Thermus thermophilus HB27 are reported. The addition of Zn2+ to the crystallization buffer was essential in order to obtain crystals. The crystals belonged to one of the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = b = 113, c = 197 Å. Diffraction data were obtained to a resolution of 2.97 Å.
PMCID: PMC2765889  PMID: 19851010
mannosyl-3-phosphoglycerate synthase; Thermus thermophilus HB27
24.  Towards Enhanced Galactose Utilization by Lactococcus lactis▿  
Applied and Environmental Microbiology  2010;76(21):7048-7060.
Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.
PMCID: PMC2976262  PMID: 20817811
25.  Statistical Inference Methods for Sparse Biological Time Series Data 
BMC Systems Biology  2011;5:57.
Comparing metabolic profiles under different biological perturbations has become a powerful approach to investigating the functioning of cells. The profiles can be taken as single snapshots of a system, but more information is gained if they are measured longitudinally over time. The results are short time series consisting of relatively sparse data that cannot be analyzed effectively with standard time series techniques, such as autocorrelation and frequency domain methods. In this work, we study longitudinal time series profiles of glucose consumption in the yeast Saccharomyces cerevisiae under different temperatures and preconditioning regimens, which we obtained with methods of in vivo nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis we first fit several nonlinear mixed effect regression models to the longitudinal profiles and then used an ANOVA likelihood ratio method in order to test for significant differences between the profiles.
The proposed methods are capable of distinguishing metabolic time trends resulting from different treatments and associate significance levels to these differences. Among several nonlinear mixed-effects regression models tested, a three-parameter logistic function represents the data with highest accuracy. ANOVA and likelihood ratio tests suggest that there are significant differences between the glucose consumption rate profiles for cells that had been--or had not been--preconditioned by heat during growth. Furthermore, pair-wise t-tests reveal significant differences in the longitudinal profiles for glucose consumption rates between optimal conditions and heat stress, optimal and recovery conditions, and heat stress and recovery conditions (p-values <0.0001).
We have developed a nonlinear mixed effects model that is appropriate for the analysis of sparse metabolic and physiological time profiles. The model permits sound statistical inference procedures, based on ANOVA likelihood ratio tests, for testing the significance of differences between short time course data under different biological perturbations.
PMCID: PMC3114728  PMID: 21518445

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