Changes in the metabolism of the microalga Chlorella vulgaris when coimmobilized in alginate with the nitrogen-fixing Phyllobacterium myrsinacearum

In an agroindustrial wastewater pond, a naturally occurring unicellular microalga, Chlorella vulgaris, was closely associated with the terrestrial plant-associative N2-fixing bacterium Phyllobacterium myrsinacearum. When the two microorganisms were artificially coimmobilized in alginate beads, they shared the same internal bead cavities, and the production of five microalgal pigments increased, but there were no effects on the number of the cells or the biomass of the microalga. The association, however, reduces the ability of C. vulgaris to remove ammonium ions and phosphorus from water. The bacterium produced nitrate from ammonium in synthetic wastewater with or without the presence of the microalga, and fixed nitrogen in two culture media. Our results suggest that interactions between microalgae and associative bacteria should be considered when cultivating microalgae for wastewater treatment.     

小球藻细胞壁缺陷型突变体的筛选及转化系统的建立

由于高效、稳定遗传转化系统的缺乏,小球藻遗传改良以及油脂代谢机理的研究等工作难以进行。研究旨在通过筛选获得小球藻Chlorella vulgaris细胞壁缺陷型突变体,在此基础上建立其转化系统。首先通过紫外诱变获得小球藻Chlorella vulgaris的突变体库,基于细胞壁缺陷型突变体在1% Triton X-100处理后叶绿素会释放到上清中的原理,利用酶标仪高通量地从约4000个突变体中筛选获得10株细胞壁缺陷的小球藻。同时以小球藻内源性-tubulin的启动子和终止子作为启动子和终止子,以AphⅧ (Aminoglycoside 3'-Phosphotransferase type Ⅷ)作为报告基因构建了转化载体pHK203。通过优化电转缓冲液组分和电击参数,确定了细胞壁缺陷型突变体CWD-3的最佳转化条件,即2 g pHK203,ddH2O作为电转缓冲液,1500 V,525 ,50 F的电击条件下,转化效率可达到40个转化子/g DNA。研究为小球藻Chlorella vulgaris的油脂代谢通路和遗传改良提供了技术基础,同时由于可降低破壁成本,筛选获得的细胞壁缺陷型突变体适于工业化生产小球藻藻粉。     

Gene expression analysis of the mechanism of inhibition of Desulfovibrio vulgaris Hildenborough by nitrate-reducing, sulfide-oxidizing bacteria

Sulfate-reducing bacteria (SRB) are inhibited by nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) in the presence of nitrate. This inhibition has been attributed either to an increase in redox potential or to production of nitrite by the NR-SOB. Nitrite specifically inhibits the final step in the sulfate reduction pathway. When the NR-SOB Thiomicrospira sp. strain CVO was added to mid-log phase cultures of the SRB Desulfovibrio vulgaris Hildenborough in the presence of nitrate, sulfate reduction was inhibited. Strain CVO reduced nitrate and oxidized sulfide, with transient production of nitrite. Sulfate reduction by D. vulgaris resumed once nitrite was depleted. A DNA macroarray with open reading frames encoding enzymes involved in energy metabolism of D. vulgaris was used to study the effects of NR-SOB on gene expression. Shortly following addition of strain CVO, D. vulgaris genes for cytochrome c nitrite reductase and hybrid cluster proteins Hcp1 and Hcp2 were upregulated. Genes for sulfate reduction enzymes, except those for dissimilatory sulfite reductase, were downregulated. Genes for the membrane-bound electron transferring complexes QmoABC and DsrMKJOP were downregulated and unaffected, respectively, whereas direct addition of nitrite downregulated both operons. Overall the gene expression response of D. vulgaris upon exposure to strain CVO and nitrate resembled that observed upon direct addition of nitrite, indicating that inhibition of SRB is primarily due to nitrite production by NR-SOB.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

The methylation-free status of a housekeeping transgene is lost at high copy number

Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.     

Copper-induced oxidative stress in the chlorophycean microalga Chlorella vulgaris: response of the antioxidant system

A concentration dependent increase in lipid peroxidation, carotenoid content and activity of superoxide dismutase was observed in the green microalga Chlorella vulgaris following copper exposure. In contrast, activities of catalase, ascorbate peroxidase and glutathione reductase, and the cellular GSH, ascorbate and K+ pool depicted a reverse trend. However, a significant rise in intracellular proline content was also evident in copper supplemented cultures. Though this study depicted the malfunction of the major antioxidant system of C. vulgaris under copper stress the test organism was found to survive and grow even at 3.0 microg mL(-1) of Cu treatment (32% growth). Further study is needed to establish the role of proline in metal toxicity regulation.     

Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI₁CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.Abbreviations Adh Alcohol dehydrogenase-1 - CaMV35S Cauliflower mosaic virus promoter - CAT Chloramphenicol acetyltransferase - CAP Chloramphenicol - AcCAP Acetylated chloramphenicol derivatives - Dicamba 3,6-dichloro-2-methoxybenzoic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T. 2,4,5-trichlorophenoxyacetic acid NRCC No. 30706     

Salicylate biodegradation by various algal-bacterial consortia under photosynthetic oxygenation

Four green microalgae (Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus and Selenastrum capricornutum), a wild Bolivian microalga strain and two cyanobacteria (Anabaena catenula and Microcystis aeruginosa) were compared for tolerance to salicylate, O2 production capacity and ability to support salicylate degradation by a Ralstonia basilensis strain in symbiotic microcosms with the microalgae. Microcystis aeruginosa had the highest tolerance to salicylate at 500 mg l(-1) and 1500 mg l(-1) but only produced 0.7 mg O2 l(-1) h(-1) in the absence of pollutant. Chlorella sorokiniana resisted salicylate at 1500 mg l(-1) with the highest O2 production in the absence of salicylate (26 mg l(-1) h(-1)) closely followed by the Bolivian microalga (23 mg l(-1) h(-1)) and Chlorella vulgaris (21 mg l(-1) h(-1)). Selenastrum capricornutum and Anabaena catenula were completely inhibited by salicylate at 500 mg l(-1). When inoculated with Ralstonia sp. and supplied with salicylate, Chlorella sorokiniana had the highest removal rate (19 mg l(-1) h(-1)), followed by the wild Bolivian strain (18 mg l(-1) h(-1)) and Chlorella vulgaris (14 mg l(-1) h(-1)).     

Tumor Suppressor p53 Down-Regulates Tissue-Specific Expression of Tyrosinase Gene in Human Melanoma Cell Lines

Tyrosinase, the key gene in melanin pigment synthesis, is tissue-specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein on tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue-specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down-regulates the tissue-specific expression of tyrosinase gene and subsequent melanin synthesis.     

Strong suppression of chloramphenicol acetyltransferase expression by inducing T7 RNA polymerase

Summary Suppression of gene expression can be used as a way to study gene regulation. Expression of chloramphenicol acetyltransferase (CAT) underE. coli promoter was inhibited by 92 % by induction of T7 RNA polymerase which transcribes from a T7 promoter opposingE. coli promoter.     

Identification of a promoter element located upstream from the hepatitis B virus X gene.

We have analyzed a series of plasmids in which the sequences located upstream from the hepatitis B virus (HBV) X gene were linked to the chloramphenicol acetyl transferase (CAT) gene. Expression of the marker CAT gene in transfected cells clearly demonstrated that sequences preceding the X gene contain an active promoter. RNA mapping by primer extension indicated that the RNA encoded by the X gene promoter initiates at multiple sites spanning nucleotides 1250 to 1350 on the HBV genome. Deletion within the adjacent HBV enhancer element region significantly reduced the activity of the X gene promoter, suggesting that the X gene promoter requires the enhancer element for maximal activity.     

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.