Phylogenetic and biochemical evidence for sterol synthesis in the bacterium Gemmata obscuriglobus

Sterol biosynthesis is viewed primarily as a eukaryotic process, and the frequency of its occurrence in bacteria has long been a subject of controversy. Two enzymes, squalene monooxygenase and oxidosqualene cyclase, are the minimum necessary for initial biosynthesis of sterols from squalene. In this work, 19 protein gene sequences for eukaryotic squalene monooxygenase and 12 protein gene sequences for eukaryotic oxidosqualene cyclase were compared with all available complete and partial prokaryotic genomes. The only unequivocal matches for a sterol biosynthetic pathway were in the proteobacterium, Methylococcus capsulatus, in which sterol biosynthesis is known, and in the planctomycete, Gemmata obscuriglobus. The latter species contains the most abbreviated sterol pathway yet identified in any organism. Analysis shows that the major sterols in Gemmata are lanosterol and its uncommon isomer, parkeol. There are no subsequent modifications of these products. In bacteria, the sterol biosynthesis genes occupy a contiguous coding region and possibly comprise a single operon. Phylogenetic trees constructed for both enzymes show that the sterol pathway in bacteria and eukaryotes has a common ancestry. It is likely that this contiguous reading frame was exchanged between bacteria and early eukaryotes via lateral gene transfer or endosymbiotic events. The primitive sterols produced by Gemmata suggest that this genus could retain the most ancient remnants of the sterol biosynthetic pathway.     

From the Cover: Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus

Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell.     

Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus

The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes. The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch. The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space. Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary. The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure.     

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.     

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.     

失血性休克大鼠L-选择素转录、翻译水平表达的研究

目的 探讨失血性休克大鼠L－选择素转录及翻译水平表达对创伤及休克病理生理过程的影响。方法 大鼠剪尾取血作为正常对照,颈部去皮颈动脉结扎剪断插管作为实验对照,建立失血性休克动物模型;遂将其分为正常对照组、实验对照组（创伤组）和实验休克组（失血性休克组）,每组均为8只。用单克隆抗体标记,流式细胞仪检测大鼠失血休克急性期中性粒细胞表面L－选择素的动态表达,以及用半定量逆转录聚合酶链反应（RT－PCR）检测大鼠白细胞胞质L－选择素mRNA半定量变化。结果 正常对照组大鼠中性粒细胞表面不同时间点L－选择素表达量（平均荧光道数）之间的差异无显著性（P＞0.05)。与正常对照组相比,实验对照组细胞表面及胞质mRNA表达均上调,细胞表面3h达峰值,4、5h持续高水平;mRNA进行性升高,于5h达峰值。与正常对照组比,实验休克组两者亦均上调;但与创伤组比,3h后则呈下调趋势。结论 创伤后中性粒细胞L－选择素表达上调,增强白细胞－内皮细胞黏附反应,有助于局部创伤愈合,修复并抵抗感染。休克组L－选择素表达下调,可减少白细胞－内皮细胞过度黏附及嵌塞毛细血管,有助于机体疏通微循环,改善灌流量。     

Peritubular cells influence Sertoli cells at the level of translation

Conditioned medium from cultured peritubular cells (PTCM) was capable of increasing the incorporation of amino acids into acid-precipitable material in cultured Sertoli cells, while the incorporation of uridine into acid-precipitable material was unaffected. PTCM did not influence intracellular cAMP accumulation in a manner similar to follicle-stimulating hormone (FSH). PTCM was able to stimulate androgen-binding protein (ABP) secretion by Sertoli cells even in the presence of a maximal dose of FSH. PTCM increased the rate at which peptides are elongated 5-fold over control medium or medium from control fibroblasts. These studies indicate that peritubular cells influence Sertoli cells through different mechanisms than FSH and exert their influence, at least in part, at the level of translation by increasing the rate of peptide elongation.     

Chromosomes in living Escherichia coli cells are segregated into domains of supercoiling.

Torsional tension in the DNA double helix can be detected in living cells of Escherichia coli from measurements of the rate of trimethylpsoralen photobinding to the intracellular DNA. Here we show that this tension is relaxed in vivo when single-strand DNA breaks are introduced by gamma-irradiation and that approximately 160 nicks per genome equivalent of DNA are required to relax greater than 95% of the tension. Chromosomes containing less than 160 nicks per genome equivalent lose only a part of the tension, depending on the number of nicks. The remaining tension is maintained during incubations of cells at 0 degrees C. Chromosomes with tension relaxed by incubation of cells with inhibitors of DNA gyrase interact with the trimethylpsoralen probe independently of the number of nicks introduced by gamma-irradiation. The results fit a model in which the chromosome in growing E. coli cells (mean generation time, 30 min) is segregated into 43 +/- 10 domains of supercoiling per genome equivalent of DNA or 120 +/- 30 domains per nucleoid. The number of domains is unchanged in cells depleted of nascent RNA by growth with rifampicin, but varies somewhat in cells growing at different rates in different media.