Whole-genome sequences of four Mycobacterium bovis BCG vaccine strains

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only vaccine available against tuberculosis (TB). A number of BCG strains are in use, and they exhibit biochemical and genetic differences. We report the genome sequences of four BCG strains representing different lineages, which will help to design more effective TB vaccines.     

Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae

NAD⁺ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD⁺ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD⁺ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD⁺-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD⁺ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD⁺ precursors. Together, our studies provide a molecular basis for how NAD⁺ homeostasis factors confer metabolic flexibility.The pyridine nucleotide NAD⁺ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD⁺ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (1, 2). NAD⁺ also plays an important role in calorie restriction (CR)²-mediated life span extension via regulating NAD⁺-dependent longevity factors (3, 4). CR is the most effective regimen known to extend life span in various species (5, 6). CR also ameliorates many age-related diseases such as cancer and diabetes (5). The Sir2 family proteins are NAD⁺-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (3, 7) and higher eukaryotes (8, 9). By coupling the cleavage of NAD⁺ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (8–10), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging.In eukaryotes, NAD⁺ is generated by de novo synthesis and by salvaging various intermediary precursors (see Fig. 1A). In yeast, the de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (11). Because the de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD⁺ precursors for the nicotinamide (Nam) moiety (11). Yeast cells also salvage Nam from NAD⁺ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD⁺ via Nma1/2 and Qns1 (see Fig. 1A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (12, 13), and Qns1 is a glutamine-dependent NAD⁺ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD⁺ auxotrophic mutants (14–16). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (14, 16). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (15, 16).Open in a separate windowFIGURE 1.Nicotinamide riboside (NmR) is an endogenous metabolite in yeast. A, the current model of the NAD⁺ biosynthesis pathways. Extracellular NmR enters the salvage cycle through Nrk1, Urh1, Pnp1, and Meu1. B, NAD⁺ prototrophic cells release metabolites into growth medium to cross-feed NAD⁺ auxotrophic cells (the npt1Δqpt1Δ and qns1Δ mutants). Micro-colonies of the NAD⁺ auxotrophic mutants become visible after 2-day incubation at 30 °C, which show “gradient” growth patterns descending from the side adjacent to WT. C, Nrk1 is required for NAD⁺ auxotrophic cells to utilize NmR. Anaerobic growth conditions (−O₂) are utilized to block de novo NAD⁺ biosynthesis in the npt1Δ and npt1Δnrk1Δ mutants. D, Nrk1 is required to utilize cross-feeding metabolites. E, cross-feeding activity is modulated by factors in NmR metabolism. Cells defective in NmR utilization (left panel) or transport (middle panel) show increased cross-feeding in spot assays. Overexpressing Nrk1 decreases cross-feeding activity (right panel). The results show growth of the npt1Δqpt1Δ recipient (plated on YPD at a density of ∼9000 cells/cm²) supported by feeder cells (∼2 × 10⁴ cells spotted directly onto the recipient lawn). oe, overexpression.NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (17). For example, NmR protected neurons from axonal degeneration via functioning as a NAD⁺ precursor (18, 19). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (14, 15, 20–22), NmR has been speculated to be an endogenous NAD⁺ precursor (17, 23). Here, we provided direct evidence for endogenous NmR as an integral part of NAD⁺ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD⁺ levels in mammalian cells (24). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes.     

Properties of a small basic Peptide from pumpkin seeds

A small basic peptide with an unusual amino acid composition has been isolated from the seeds of pumpkin, Cucurbita maxima. Amino acid analysis and sequence data show the protein to be about 36 residues in length, with an approximate composition Lys₁, Arg₁₄, Asp₃, (Glu + Gln)₁₅, Gly₁, Pro₁, Trp₁. On the basis of composition, the molecular weight is approximately 5000 daltons and the nitrogen content by weight is 20.4%. Twelve amino acids are entirely lacking. The peptide is slightly toxic to mouse B-16 melanoma cells, but its in vivo function is unknown. It does not appear to be derived from cucurbitin, the pumpkin storage globulin; however, it could be a storage peptide involved in nitrogen mobilization during the early stages of germination.     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.     

Vegetation and soil properties in restored wetlands near Lake Taihu,China

Riparian wetlands are important components of the lake ecosystem, and they play essential roles in maintaining system health. Remediation of degraded lakeshore wetlands is an essential component of lake restoration. A study was conducted to investigate the restoration of lakeshore wetlands, which were converted to rice fields and then abandoned for 2, 5, 10 and 15 years, near Lake Taihu. Soil samples (0–20 cm and 20–40 cm) were taken and plant species were investigated. The carbon content in the soil had increased significantly, rising from 0.71% to 1.85% between 2 and 15 years. Organic matter accumulation improved soil texture, and water stable aggregate content (>0.25 mm) and soil porosity increased. Total nitrogen in the soil increased from 0.06% to 0.13%, and total Kjeldahl nitrogen increased from 124.4 mg kg⁻¹ to 351.5 mg kg⁻¹. Total phosphorus in the soil increased from 0.045% to 0.071%, and the Olsen-P value increased from 5.13 mg kg⁻¹ to 16.0 mg kg⁻¹. Results showed that phosphorous did not increase as much as nitrogen. In the vegetation restoration process, plant species composition moved towards a natural wetland community, and spatial heterogeneity and landscape diversity increased. The richness of plant biodiversity increased rapidly in the first 2 years, then more slowly in later restoration stages. The wetlands recovery process may be complicated by interactions of biota and soil and hydrological conditions.     

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or “rack‐induced” state for ET functions. We herein report circular permutation of azurin by linking the N‐ and C‐termini and creating new termini in the loops between 1^st and 2^nd β strands or between 3^rd and 4^th β strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogen‐bonding network in secondary coordination sphere around the T1Cu center.     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.