Metagenomic gene discovery: How far have we moved into novel sequence space?

Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment. The advent of the technology generated great excitement, as it has provided new opportunities and technologies for studying the wealth of microbial genetic diversity in the environment. Metagenomics has been widely predicted to access new dimensions of protein sequence space. A decade on, we review how far we have actually moved into new sequence space (and other aspects of protein space) using metagenomic tools. While several novel enzyme activities and protein structures have been identified through metagenomic strategies, the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. This is particularly true for glycosyl hydrolases and lipase/esterases, despite the fact that these activities are frequently screened for in metagenomic studies. However, there is much room for improvement in the methods employed and they will need to be addressed so that access to novel biocatalytic activities can be widened.     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT-PCR和PCR-RACE技术,从菊科植物甘菊(Dendranthema lavandulifolium)中克隆到2个甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821 bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918 bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97%,推导的氨基酸序列的同源性为98%。与已发表的其它植物BADH基因氨基酸序列的同源性在64%以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C)。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT-PCR-Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries

ABSTRACT. Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray–Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.     

Inhibition of Aβ42 aggregation using peptides selected from combinatorial libraries

Increasing evidence suggests that the aggregation of the small peptide Aβ42 plays an important role in the development of Alzheimer's disease. Inhibiting the initial aggregation of Aβ42 may be an effective treatment for preventing, or slowing, the onset of the disease. Using an in vivo screen based on the enzyme EGFP, we have searched through two combinatorially diverse peptide libraries to identify peptides capable of inhibiting Aβ42 aggregation. From this initial screen, three candidate peptides were selected and characterized. ThT studies indicated that the selected peptides were capable of inhibiting amyloid aggregation. Additional ThT studies showed that one of the selected peptides was capable of disaggregating preformed Aβ42 fibers. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system

Aims: The aim of the present work was to characterize the heterotrophic bacterial community of a marine recirculating aquaculture system (RAS). Methods and Results: An experimental RAS was sampled for the rearing water (RW) and inside the biofilter. Samples were analysed for bacterial abundances, community structure and composition by using a combination of culture‐dependent and ‐independent techniques. The most represented species detected among biofilter clones was Pseudomonas stutzeri, while Ruegeria spp. and Roseobacter spp. were more abundant among isolates. In comparison, the genera Roseobacter and Ruegeria were well represented in both the biofilter and the RW samples. A variety of possible bacterial pathogens (e.g. Vibrio spp., Erwinia spp. and Coxiella spp.) were also identified in this study. Conclusions: Results revealed that the bacterial community in the RW was quite different to that associated with the biofilter. Moreover, data obtained suggest that the whole bacterial community can be involved in maintaining an effective and a stable rearing environment (shelter effect). Significance and Impact of the Study: Improving the reliability and the sustainability of RAS depends on the correct management of the bacterial populations inside it. This study furnishes more accurate information on the bacterial populations and better clarifies the existing relationships between the bacterial flora in the RW and that associated with the biofilter.     

粗毛栓菌cDNA文库的构建和漆酶基因的表达分析

粗毛栓菌（Trametes gallica）能够分泌多种胞外氧化酶并且快速降解木质纤维素.为了快速高效分离鉴定粗毛栓菌木质纤维素降解酶相关基因,用Trizol试剂提取不同培养条件下粗毛栓菌总RNA,用Creator^TM SMART^TM cDNA Library Construction Kit和Advantage^®2 PCR Kit成功构建了该菌全长cDNA文库.原始文库滴度为1.5×10⁵cfu,重组率达99%,插入片段在0.7～2.0 kb之间,平均大小约1 kb. 随机取16个重组子进行测序,全长cDNA序列完整性率为85.7%;并筛选到1个漆酶基因,编码区长1 551 bp,预测的蛋白质由517个氨基酸残基组成,分子量为55.41 kD,等电点为4.76.用半定量RT-PCR法分析了该漆酶基因在不同培养条件下的表达水平. 结果显示,高浓度的碳源,氮源,Cu^2＋均能诱导此基因的表达,该结果为漆酶基因的表达调控机制的深入研究奠定了基础.