Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of K_m 276 μM, k_cat 1.26 min⁻¹ and k_cat/K_m 76.2 M⁻¹ s⁻¹. These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.     

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.     

Isolated Skull Cryptococcosis in an Immunocompetent Patient

A 62-year-old immunocompetent rural woman who represents an isolated cryptococcal skull infection without systematic involvement is described. Diagnosis was based on positive India ink staining, positive histopathologic examination, and positive culture. Species identification was performed by growth on Sabouraud dextrose agar and CHROMagar medium and by sequencing of the intergenic and internal transcribed spacer regions of the rRNA genes. This case describes a rare presentation of Cryptococcus neoformans infection in a human immunodeficiency virus-negative patient. The lesions were significantly improved with treatment of daily oral itraconazole 400 mg. A maintenance therapy with a low-dose itraconazole was prescribed to warrant a clinical and mycological eradication. A two-year follow-up did not show any recurrence of infection.     

Locating QTLs controlling several adult root traits in an elite Chinese hybrid rice

This study aimed to elucidate the genetics of the adult root system in elite Chinese hybrid rice. Several adult root traits in a recombinant inbred line (RIL) population of Xieyou 9308 and two backcross F₁ (BCF₁) populations derived from the RILs were phenotyped under hydroponic culture at heading stage for quantitative trait locus (QTL) mapping and other statistical analysis. There a total of eight QTLs detected for the root traits. Among of them, a pleiotropic QTL was repeatedly flanked by RM180 and RM5436 on the short arm of chromosome 7 for multiple traits across RILs and its BCF₁ populations, accounting for 6.88% to 25.26% of the phenotypic variances. Only additive/dominant QTLs were detected for the root traits. These results can serve as a foundation for facilitating future cloning and molecular breeding.     

Analysis of B-Class Genes NAP3L3 and NAP3L4 in Narcissus tazetta var. chinensis

Very few flower organ identity genes have been characterized in Chinese narcissus (Narcissus tazetta var. chinensis), which has petaloid sepals. Here, we report the cloning of two full-length B-class genes, namely NAP3L3 and NAP3L4, that are orthologs of the DEFICIENS lineage. Both genes are highly expressed in the second whorl of the perianth and in the stamens. NAP3L4 is also expressed strongly in the ovule. The functions of these two genes were further analyzed using transgenic plants. Ectopic expression of either gene in Arabidopsis gave no obvious floral organ transformation phenotypes. In yeast two-hybrid assays, NAP3L3 and NAP3L4 failed to homodimerize and interacted weakly with each other. The data suggest that these two genes might not be involved in the formation of petaloid sepals. Isolation and functional analysis of other B-class paralogs should be conducted to fully understand petaloid tepal development in Chinese narcissus.     

Effect of Selective Substitution of 5-Bromocytosine on Conformation of DNA Triple Helices

Abstract

Three triplex DNAs containing 5-bromocytosine[^BrC] were studied by vibrational spectroscopy and molecular modelling. Firstly, three oligodeoxypyrimidines of 5′-(TC)3- T4-(^BrCT)3 [C^BrC], 5′-(T^BrC)3-T4-(CT)3 pCC] and 5′-(T^BC)3-T4-(^BrCT)3 [^BrC^BC] were synthesized and then reacted with an oligodeoxypurine of 5′-(AG)₃ at pH=4.5 in phosphate buffer respectively to form three comparative hairpin triplex named CY,YC and YY. The results of FT-Raman and IR revealed that YY is almost in A-like form, CY and YC are combinations of A-like form and B-like form, but A-form dominates in CY while B-form is equivalent as A-form in YC. The result is consistent with the theoretical analysis.     

Removal of Contaminating Metals from Soil by Sulfur-Based Bioleaching and Biogenic Sulfide-Based Precipitation

The potential of a hybrid process incorporating sulfur-based bioleaching and sulfide-based precipitation for treatment of metal-contaminated soil was examined in batch-type experiments. The sulfur-based soil bioleaching process with Acidithiobacillus sp. could be initiated at a wide range of initial pH from 4.0 to 6.3. After 15 days, 98% of Zn, 89% of Cu and 79% of Cd was bioleached. The gaseous sulfides recycling from Desulfovibrio sp.-mediated sulfate-reducing reactor via N₂ sparging efficiently treated metal-loaded soil leachate. With a sulfide/metal ratio of 3.0, 88% of Zn, 100% of Cu and 95% of Cd were precipitated, resulting in effluent metal concentrations of 3.5 mg Zn²⁺/L, 0.2 mg Cu²⁺/L and 0.03 mg Cd²⁺/L.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.