Pyrobaculum yellowstonensis Strain WP30 Respires on Elemental Sulfur and/or Arsenate in Circumneutral Sulfidic Geothermal Sediments of Yellowstone National Park

Thermoproteales (phylum Crenarchaeota) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph''s Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 μM As) and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.     

Cloning of ε‐poly‐L‐lysine (ε‐PL) synthetase gene from a newly isolated ε‐PL‐producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans

ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l⁻¹ when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24–30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis.     

Genome-wide gene-environment study identifies glutamate receptor gene GRIN2A as a Parkinson's disease modifier gene via interaction with coffee

Our aim was to identify genes that influence the inverse association of coffee with the risk of developing Parkinson''s disease (PD). We used genome-wide genotype data and lifetime caffeinated-coffee-consumption data on 1,458 persons with PD and 931 without PD from the NeuroGenetics Research Consortium (NGRC), and we performed a genome-wide association and interaction study (GWAIS), testing each SNP''s main-effect plus its interaction with coffee, adjusting for sex, age, and two principal components. We then stratified subjects as heavy or light coffee-drinkers and performed genome-wide association study (GWAS) in each group. We replicated the most significant SNP. Finally, we imputed the NGRC dataset, increasing genomic coverage to examine the region of interest in detail. The primary analyses (GWAIS, GWAS, Replication) were performed using genotyped data. In GWAIS, the most significant signal came from rs4998386 and the neighboring SNPs in GRIN2A. GRIN2A encodes an NMDA-glutamate-receptor subunit and regulates excitatory neurotransmission in the brain. Achieving P_2df = 10⁻⁶, GRIN2A surpassed all known PD susceptibility genes in significance in the GWAIS. In stratified GWAS, the GRIN2A signal was present in heavy coffee-drinkers (OR = 0.43; P = 6×10⁻⁷) but not in light coffee-drinkers. The a priori Replication hypothesis that “Among heavy coffee-drinkers, rs4998386_T carriers have lower PD risk than rs4998386_CC carriers” was confirmed: OR_Replication = 0.59, P_Replication = 10⁻³; OR_Pooled = 0.51, P_Pooled = 7×10⁻⁸. Compared to light coffee-drinkers with rs4998386_CC genotype, heavy coffee-drinkers with rs4998386_CC genotype had 18% lower risk (P = 3×10⁻³), whereas heavy coffee-drinkers with rs4998386_TC genotype had 59% lower risk (P = 6×10⁻¹³). Imputation revealed a block of SNPs that achieved P_2df<5×10⁻⁸ in GWAIS, and OR = 0.41, P = 3×10⁻⁸ in heavy coffee-drinkers. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in GWAS. Both adenosine antagonists (caffeine-like) and glutamate antagonists (GRIN2A-related) are being tested in clinical trials for treatment of PD. GRIN2A may be a useful pharmacogenetic marker for subdividing individuals in clinical trials to determine which medications might work best for which patients.     

Incipient Genome Differentiation in Gossypium. III. Comparison of Chromosomes of G. HIRSUTUM and Asiatic Diploids Using Heterozygous Translocations

Hybrids between upland cotton (G. hirsutum, genome constitution 2A_hD_h) and either A-genome or D-genome diploid species exhibit 26 paired and 13 unpaired chromosomes at metaphase I. The A_h and D_h genomes are therefore considered homoeologous with those of the respective diploids. Previous studies, nevertheless, revealed a low level of ("incipient") differentiation between D_h and various diploid D genomes. The diploid A genomes have been regarded as more closely homologous to A_h on the basis of low preferential pairing and autotetraploid segregation ratios in allohexaploids.—The present study addressed the following questions: Are the diploid A genomes differentiated from A_h in meiotic homology? If so, is the differentiation manifested equally by all 13 chromosomes or is it localized in certain chromosomes?—Three diploid A-genome lines representing G. herbaceum and G. arboreum were hybridized by in ovulo culture of embryos (1) with a standard line of G. hirsutum, which differs from G. herbaceum by two and from G. arboreum by three naturally occurring reciprocal translocations involving chromosomes 1–5, and (2) with six lines homozygous for experimental translocations involving chromosomes 6, 7, 10, 11, 12 and 13. Chiasma frequencies in hybrids were compared with those in appropriate G. hirsutum controls. In every comparison overall chiasma frequencies were slightly lower in the hybrids. Therefore A_h appears to be differentiated from the diploid A genomes. No localized differentiation was detected in chromosomes marked by experimental translocations. The differentiation may be localized mainly in chromosomes 4 and 5.     

Contributions of leaf photosynthetic capacity, leaf angle and self-shading to the maximization of net photosynthesis in Acer saccharum: a modelling assessment

Background and Aims

Plants are expected to maximize their net photosynthetic gains and efficiently use available resources, but the fundamental principles governing trade-offs in suites of traits related to resource-use optimization remain uncertain. This study investigated whether Acer saccharum (sugar maple) saplings could maximize their net photosynthetic gains through a combination of crown structure and foliar characteristics that let all leaves maximize their photosynthetic light-use efficiency (ɛ).

Methods

A functional–structural model, LIGNUM, was used to simulate individuals of different leaf area index (LAI_ind) together with a genetic algorithm to find distributions of leaf angle (L_A) and leaf photosynthetic capacity (A_max) that maximized net carbon gain at the whole-plant level. Saplings grown in either the open or in a forest gap were simulated with A_max either unconstrained or constrained to an upper value consistent with reported values for A_max in A. saccharum.

Key Results

It was found that total net photosynthetic gain was highest when whole-plant PPFD absorption and leaf ɛ were simultaneously maximized. Maximization of ɛ required simultaneous adjustments in L_A and A_max along gradients of PPFD in the plants. When A_max was constrained to a maximum, plants growing in the open maximized their PPFD absorption but not ɛ because PPFD incident on leaves was higher than the PPFD at which ɛ_max was attainable. Average leaf ɛ in constrained plants nonetheless improved with increasing LAI_ind because of an increase in self-shading.

Conclusions

It is concluded that there are selective pressures for plants to simultaneously maximize both PPFD absorption at the scale of the whole individual and ɛ at the scale of leaves, which requires a highly integrated response between L_A, A_max and LAI_ind. The results also suggest that to maximize ɛ plants have evolved mechanisms that co-ordinate the L_A and A_max of individual leaves with PPFD availability.     

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha

The NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD⁺, as well as reverse electron transfer from NADH to H⁺, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H⁺/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD⁺ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD⁺/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD⁺, respectively. Catalysis in both directions is product inhibited with K _I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.     

Female-to-Male Breeding Ratio in Modern Humans—an Analysis Based on Historical Recombinations

Was the past genetic contribution of women and men to the current human population equal? Was polygyny (excess of breeding women) present among hominid lineages? We addressed these questions by measuring the ratio of population recombination rates between the X chromosome and the autosomes, ρ_X/ρ_A. The X chromosome recombines only in female meiosis, whereas autosomes undergo crossovers in both sexes; thus, ρ_X/ρ_A reflects the female-to-male breeding ratio, β. We estimated β from ρ_X/ρ_A inferred from genomic diversity data and calibrated with recombination rates derived from pedigree data. For the HapMap populations, we obtained β of 1.4 in the Yoruba from West Africa, 1.3 in Europeans, and 1.1 in East Asian samples. These values are consistent with a high prevalence of monogamy and limited polygyny in human populations. More mutations occur during male meiosis as compared to female meiosis at the rate ratio referred to as α. We show that at α ≠ 1, the divergence rates and genetic diversities of the X chromosome relative to the autosomes are complex functions of both α and β, making their independent estimation difficult. Because our estimator of β does not require any knowledge of the mutation rates, our approach should allow us to dissociate the effects of α and β on the genetic diversity and divergence rate ratios of the sex chromosomes to the autosomes.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K_m^app value for d-xylose was 8.6 mM and the V_max^app was 20.8 units/mg NADH was used as a coenzyme. The K_m^app values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.     

Inhibitor studies on non-photochemical plastoquinone reduction and H2 photoproduction in Chlamydomonas reinhardtii

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O₂ uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H₂ in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H₂ production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H₂ production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.     

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.     

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K_ass∼10⁶ M⁻¹) for human telomeric antiparallel quadruplex d(TTAGGG)₄ and duplex d(TTAGGG)₄∶d(CCCTAA)₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na⁺ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K⁺ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.     

Influence of Maternal Phenotype on Metabolic Differentiation of Agouti Locus Mutants in the Mouse

The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A ^vy, or mottled, a^m, mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A^vy/a and a^m/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.—In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a^m/a^m mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.—Mottled yellow A^vy/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A^vy/a siblings.—In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A^vy/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A ^vy/a dams produced significantly fewer pseudoagouti A ^vy/a offspring than did black a/a dams.—The data suggest that metabolic differentiation of A^vy/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.     

Relationship between the pregnenolone-induced difference spectrum and cytochrome b5 reduction in guinea-pig adrenal microsomes

Addition of pregnenolone to guinea-pig adrenal microsomes produces a slowly developing difference spectrum with peaks at about 425 and 557 nm and a trough at about 410 nm. The spectral change is similar to that resulting from the reduction of cytochrome b₅ by NADH or NADPH. In the presence of sufficient quantities of NADH to fully reduce cytochrome b₅, pregnenolone produces a typical type I difference spectrum (ΔA_385–420 nm). Pregnenolone is converted to progesterone by adrenal microsomes without addition of cofactor (NAD⁺) for the 3β-hydroxysteroid dehydrogenase (HSD) reaction. The rate of conversion is increased 2–3 fold by NAD⁺ and inhibited by NADH. Accompanying the metabolism of pregnenolone (with or without added NAD⁺) is the production of NADH and reduction of cytochrome b₅. Addition of pregnenolone alone to adrenal microsomes results in 60–80% reduction of cytochrome b₅. The reduction of cytochrome b₅ is maintained for at least as long as pregnenolone is being metabolized. Inhibition of pregnenolone metabolism changes the pregnenolone-induced spectral change to a type I and prevents the reduction of cytochrome b₅. The results suggest that the oxidation-reduction state of cytochrome b₅ in adrenal microsomes is controlled in part by pregnenolone metabolism which in turn influences the pregnenolone-induced difference spectrum. Oxidation of NADH by cytochrome b₅ may serve to prevent NADH inhibition of HSD activity and to generate additional NAD⁺ as cofactor for the reaction.     

Babesia bovis: Proteins of virulent and avirulent parasites passaged through ticks and splenectomized or intact calves

Passage of the avirulent vaccine (K) strain of Babesia bovis (K_A) through either Boophilus microplus ticks, intact calves, or intact calves and then ticks, resulted in two distinct protein and protein antigen profiles as analyzed by two-dimensional gel electrophoresis of biosynthetically labeled proteins and immunoprecipitates. Different degrees of expression of two major acidic antigens of K_A designated Ka₁ (M_r 47,500) and Ka₂ (M_r 43,000) were observed. Ka₁ was apparently lost following passage of K_AB. bovis through intact calves but was strongly represented in the parasite population following a single tick passage. In contrast, passage through ticks of the virulent K_VB. bovis (from which K_A was derived by passage in splenectomized calves) did not lead to strong representation of the Ka₁ protein although there was increased representation of another major acidic protein antigen, designated Kv (M_r 35,000). These data suggest that the previously recognized reversion to a strain-dependent basal antigenic type in the tick vector depends also on intrastrain characteristics such as virulence and strain heterogeneity. The data suggest that K_A is a more heterogeneous population than K_V although cloned isolates are required to establish this point. Comparable syringe passage of another strain of B. bovis, designated C strain, through splenectomized calves resulted in less marked differences between the putative C_A and C_VB. bovis. This may explain the less stable avirulence of C_A compared to K_AB. bovis. Various selection pressures must act, in either the tick or the vertebrate host, on subpopulations in heterogeneous isolates to produce the changes described in protein antigen profiles of B. bovis. The possible relevance of changes in representation of proteins to biological characteristics of B. bovis (such as virulence and tick transmissibility) is discussed.     

Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T_m) by 6–7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) ⇄ I_T(±MgATP) → A_T → (A_T)_n. The equilibrium unfolding to intermediate, I_T, is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A_T, for which aggregation to (A_T)_n and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A_T.     

Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N⁶-methyladenine (6 mA) and N⁴-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN₇TAY-3′/3′-CTN₇ ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.     

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F₉ MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.     

Causes of decreased photosynthetic rate and metabolic capacity in water-deficient leaf cells: a critical evaluation of mechanisms and integration of processes

Background

Water deficit (WD) decreases photosynthetic rate (A) via decreased stomatal conductance to CO₂ (g_s) and photosynthetic metabolic potential (A_pot). The relative importance of g_s and A_pot, and how they are affected by WD, are reviewed with respect to light intensity and to experimental approaches.

Scope and Conclusions

With progressive WD, A decreases as g_s falls. Under low light during growth and WD, A is stimulated by elevated CO₂, showing that metabolism (A_pot) is not impaired, but at high light A is not stimulated, showing inhibition. At a given intercellular CO₂ concentration (C_i) A decreases, showing impaired metabolism (A_pot). The C_i and probably chloroplast CO₂ concentration (C_c), decreases and then increases, together with the equilibrium CO₂ concentration, with greater WD. Estimation of C_c and internal (mesophyll) conductance (g_i) is considered uncertain. Photosystem activity is unaffected until very severe WD, maintaining electron (e⁻) transport (ET) and reductant content. Low A, together with photorespiration (PR), which is maintained or decreased, provides a smaller sink for e⁻_, causing over-energization of energy transduction. Despite increased non-photochemical quenching (NPQ), excess energy and e⁻ result in generation of reactive oxygen species (ROS). Evidence is considered that ROS damages ATP synthase so that ATP content decreases progressively with WD. Decreased ATP limits RuBP production by the Calvin cycle and thus A_pot. Rubisco activity is unlikely to determine A_pot. Sucrose synthesis is limited by lack of substrate and impaired enzyme regulation. With WD, PR decreases relative to light respiration (R_L), and mitochondria consume reductant and synthesise ATP. With progressing WD at low A, R_L increases C_i and C_c. This review emphasises the effects of light intensity, considers techniques, and develops a qualitative model of photosynthetic metabolism under WD that explains many observations: testable hypotheses are suggested.Key words: Water stress, photosynthesis, photorespiration, stomata, ATP synthase, ATP, photoinhibition, electron transport, Rubisco, fluorescence, sucrose, mesophyll conductance     

Immobilized thermophilic bacteria as a source of respiratory chain for the recycling of NAD+

Immobilization of thermophilic bacterium strain PS3 has been performed by crosslinking with 0.4% glutaraldehyde in the presence of 4.6% bovine serum albumin at ? 20°C. After immobilization of bacteria the plasma membrane became permeable to NADH. The yield of NADH respiration of the immobilized strain was ～10%. The apparent K_m for NADH with the immobilized thermophilic strain was $\text{6 × 10}{}^{\mathrm{?4}}\text{m}$. After immobilization of this strain no variations of activities were observed between pH 4.5 and 9.5. Recycling of NAD⁺ is at least 10 times better with the thermophilic strain compared to Escherichia coli. In a preliminary experiment at 45°C the half life obtained with Escherichia coli was 1 h and with PS3 was 12 h. This temperature increased the rate of respiration by a factor of ～4 (compared to 20°C) and may avoid most of bacterial contaminations (most bacteria are not able to grow above 42°C).