Ecological Genomics of Marine Roseobacters

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.     

Surface Colonization by Marine Roseobacters: Integrating Genotype and Phenotype

The Roseobacter clade is a broadly distributed, abundant, and biogeochemically relevant group of marine bacteria. Representatives are often associated with organic surfaces in disparate marine environments, suggesting that a sessile lifestyle is central to the ecology of lineage members. The importance of surface association and colonization has been demonstrated recently for select strains, and it has been hypothesized that production of antimicrobial agents, cell density-dependent regulatory mechanisms, and morphological features contribute to the colonization success of roseobacters. Drawing on these studies, insight into a broad representation of strains is facilitated by the availability of a substantial collection of genome sequences that provides a holistic view of these features among clade members. These genome data often corroborate phenotypic data but also reveal significant variation in terms of gene content and synteny among group members, even among closely related strains (congeners and conspecifics). Thus, while detailed studies of representative strains are serving as models for how roseobacters transition between planktonic and sessile lifestyles, it is becoming clear that additional studies are needed if we are to have a more comprehensive view of how these transitions occur in different lineage members. This is important if we are to understand how associations with surfaces influence metabolic activities contributing to the cycling of carbon and nutrients in the world''s oceans.The Roseobacter lineage is an abundant marine bacterial group whose members mediate key biogeochemical processes and have been the subject of several recent reviews (e.g., references 7, 11, and 91). While roseobacters are broadly distributed across diverse marine environments, roseobacter abundance is often highest near phytoplankton blooms or macroalgae, or in association with organic particles, suggesting that cell-surface interactions are a defining feature of lineage members. This is supported by culture-independent studies identifying roseobacters as ubiquitous and rapid colonizers of a variety of inorganic and organic marine surfaces, including marine algae and dinoflagellates (19, 20, 51). Though little is known of the exact mechanism(s) that roseobacters employ to physically associate with eukaryotic cell surfaces or particles, several cultivated strains have been shown to be capable of surface colonization (8, 71). Furthermore, laboratory-based studies confirm expression of traits expected to be important in colonization success, including possession of holdfast structures, motility, and chemotaxis, as well as production of quorum sensing (QS) molecules and antimicrobial metabolites in select strains. Despite the recognized importance of roseobacters in natural systems and the numerous (∼40) genome sequences that are, or are to soon be, publicly available (7), laboratory investigations of phenotypes expected to contribute to the ecological success of roseobacters are limited. Thus, a comparative genome analysis of functions likely to contribute to, or even define, different colonizing abilities can provide a more holistic view of how widely distributed and conserved these features are among diverse clade members and may facilitate hypothesis-driven research in some areas.     

Biosynthesis of d-Erythroascorbic Acid by Candida

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.     

Genetic Dissection of Histone Function

Mutational analysis is an essential tool for understanding the functions of genes within a living organism. The budding yeastSaccharomyces cerevisiaeprovides an excellent model system for dissecting the genetics of histone function at the molecular and cellular levels. A simple gene organization, plus a wide variety of genetic strategies, makes it possible to directly manipulate a specific histone genein vitroand then examine the expression of mutant allelesin vivo.Recent methods for manipulating the yeast histone genes have been designed to facilitate both site-directed analysis of structure/function relationships and unbiased screens targeted at specific functional pathways. The conservation of histone and nucleosome structure throughout evolution means that the principles discovered through genetic studies in yeast will be broadly applicable to the chromatin of more complex eukaryotes.     

心脏发育的基因调控

心脏发育是一个复杂的过程.在脊椎动物和无脊椎动物果蝇中驱动早期心脏分化的基因具有惊人的相似性.以果蝇、斑马鱼、小鼠等作为模式动物,以心脏的发育过程为主线,探讨了心脏发育的基因调控的研究进展.     

Inhibition of Phosphatidic Acid Biosynthesis by cis-9,10-Methylene Hexadecanoic Acid

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.     

Biosynthesis of jasmonic Acid by several plant species

Six plant species metabolized ¹⁸O-labeled 12-oxo-cis,cis-10,15-phytodienoic acid (12-oxo-PDA) to short chain cyclic fatty acids. The plant species were corn (Zea mays L.), eggplant (Solanum melongena L.), flax (Linum usitatissimum L.), oat (Avena sativa L.), sunflower (Helianthus annuus L.), and wheat (Triticum aestivum L.). Among the products was jasmonic acid, a natural plant constituent with growth-regulating properties. The pathway is the same as the one recently reported by us for jasmonic acid synthesis in Vicia faba L. pericarp. First, the ring double bond of 12-oxo-PDA is saturated; then β-oxidation enzymes remove six carbons from the carboxyl side chain of the ring. Substrate specificity studies indicated that neither the stereochemistry of the side chain at carbon 13 of 12-oxo-PDA nor the presence of the double bond at carbon 15 was crucial for either enzyme step. The presence of enzymes which convert 12-oxo-PDA to jasmonic acid in several plant species indicates that this may be a general metabolic pathway in plants.     

A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.     

Ecological genomics of marine Roseobacters

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.     

Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.     

Biosynthesis of Biotin-vitamers from Oleic Acid by Cryptococcus neoformans

Chitosanase (ChoA) from Mitsuaria chitosanitabida 3001 was successfully evolved with secretion efficiency and thermal stability. The inactive ChoA mutant (G151D) gene was used to mutate by an error-prone PCR technique and mutant genes that restored chitosanase activity were isolated. Two desirable mutants, designated M5S and M7T, were isolated. Two amino acids, Leu74 and Val75, in the signal peptide of ChoA were changed to Gln and Ile respectively in the M7T mutant, in addition to the G151D mutation. The L74Q/V75I double ChoA mutant was 1.5-fold higher in specific activity than wild-type ChoA due to efficient secretion of ChoA. One amino acid Asn222 was changed to Ser in the M5S mutant in addition to the G151D mutation. The N222S single ChoA mutant was 1.2-fold higher in specific activity and showed a 17% increase in thermal stability at 50 °C as compared with wild-type ChoA. This is the first study to achieve an evolutional increase in enzyme capability among chitosanses.     

Ionylideneacetic Acids and Abscisic Acid Biosynthesis by Cercospora rosicola

Several compounds having the basic α-ionylideneacetic acid structure were tested in Cercospora rosicola resuspensions. At 100 μm, all the compounds inhibited abscisic acid (ABA) biosynthesis. Time studies with unlabelled and deuterated (2Z,4E)- and (2E,4E)-α-ionylideneacetic acids showed rapid conversions into both (2Z,4E)- and (2E,4E)-4′-keto-α-ionylideneacetic acids as major products. Incorporation of the label into ABA was specific for the 2Z,4E-isomer. Minor products, identified by GC-MS, were (2Z,4E)- and (2E,4E)-4′-hydroxy-α-ionylideneacetic acids and (2Z,4E)-1′-hydroxy-α-ionylideneacetic acid. The conversion to (2Z,4E)-l′-hydroxy-α-ionylideneacetic acid has not been previously reported and was specific for the 2Z,4E-isomer. A time study for the conversion of methyl esters of [²H₃]-(2Z,4E)- and [²H₃]-(2E,4E)-4′-keto-α-ionylideneacetates showed a slow introduction of the l′-hydroxyl group and specificity for 2Z,4E-isomer. Conversion of the ethyl esters of (2Z,4E)- and (2E,4E)-l′-hydroxy-α-ionylideneacetates into the ethyl esters of both ABA and (2E,4E)-ABA demonstrated that ABA can be formed by oxidation of the 4′-position after the insertion of the 1′-hydroxy group. The ethyl 1′-hydroxy acids were also isomerized to the corresponding ethyl (2Z,4E)- and ethyl (2E,4E)-3′-hydroxy-β-ionylideneacetates. Ethyl (2Z,4E)-1′-hydroxy acid also gave small amounts of ethyl l′,4′-trans-diol of ABA. These results suggest that ABA may be formed through a (2Z,4E)-1′-hydroxy-α-ionylidene-type intermediate in addition to the previously proposed route through (2Z,4E)-4′-keto-α-ionylideneacetic acid.     

海洋细菌产生的多不饱和脂肪酸

鱼油的主要成分为二十碳五烯酸和二十二碳六烯酸,这些多不饱和脂肪酸具有多方面的生理活性。近年来在海洋细菌中发现这些多不饱和脂肪酸的存在,海洋细菌很可能是这些多不饱和脂肪酸的原始生产之一。对其生物合成的深入研究表明,海洋细菌多不饱和脂肪酸的合成不同于其他生物的不饱和脂肪酸的合成机制,合成过程中不涉及重要的脂肪酸脱氢和延长机制,其合成由一种多聚乙酰合成酶(PKS)催化。