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Lee SW Won K Lim HK Kim JC Choi GJ Cho KY 《Applied microbiology and biotechnology》2004,65(6):720-726
The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C4) but not p-nitrophenyl palmitate (C16). 相似文献
3.
碱性土壤微生物基因的克隆和多样性分析 总被引:3,自引:0,他引:3
从碱性土壤样品中直接抽提和分离宏基因组DNA, 首先构建了包含5 562个阳性克隆的碱性土壤16S rDNA文库, 随机抽取9个克隆测序后构建的系统进化树表明了碱性土壤环境微生物种群基因的多样性。纯化土壤宏基因组DNA后采用EcoRⅠ酶部分酶切处理, 我们又构建了以pGEM-3Zf(+)为载体的DNA部分文库AL01。AL01文库包含23650个克隆, 随机插入载体的外源DNA片段平均大小为3.2 kb左右, DNA文库的总容量为75.68 Mb。建库效率为从每克环境样品中获得6 000个左右的含随机外源DNA片段插入载体的克隆。采用酶活筛选策略, 我们从AL01文库中筛选到一个编号为pGXAA2011的阳性克隆携带有一个完整的碱性蛋白酶基因。蛋白酶活性检测其酶活作用最佳温度为40℃, 最适作用pH 值为9.5。另外, 我们还克隆和表达了一个新型b-葡萄糖苷酶基因unglu01, 该基因和现有数据库中的b-葡萄糖苷酶基因没有任何DNA或者氨基酸水平的同源性。将unglu01基因的ORF与表达载体pETBlue-2连接后导入宿主菌株Tuner(DE3)pLacI中, 该重组表达克隆在含柠檬酸高铁铵和七叶苷的LA平板上表现清晰的b-葡萄糖苷酶活性, SDS-PAGE电泳可以检测到29 kDa大小的目的蛋白。 相似文献
4.
《Journal of microbiological methods》2009,76(3):515-522
The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil. 相似文献
5.
Next‐generation DNA sequencing has enabled a rapid expansion in the size of molecular fungal ecology studies employing the nuclear internal transcribed spacer (ITS) region. Many sequence‐processing pipelines and protocols require sequence clustering to generate operational taxonomic units (OTUs) based on sequence similarity as a step to reduce total data quantity and complexity prior to taxonomic assignment. However, the consequences of ITS sequence clustering in regard to sample taxonomic coverage have not been carefully examined. Here we demonstrate that typically used clustering thresholds for fungal ITS sequences result in statistically significant losses in taxonomic coverage. Analyses using environmentally derived fungal sequences indicated an average of 3.1% of species went undetected (P < 0.05) if the sequences were denoised and clustered at a 97% threshold prior to taxonomic assignment. Additionally, an in silico analysis using a reference fungal ITS database suggested that approximately 25% of species went undetected if the sequences were clustered prior to taxonomic assignment. Finally, analysis of sequences derived from pure‐cultured fungal isolates of known identity indicated sequence denoising and clustering were not critical in improving identification accuracy. 相似文献
6.
Sim SJ Baik KS Park SC Choe HN Seong CN Shin TS Woo HC Cho JY Kim D 《Journal of industrial microbiology & biotechnology》2012,39(4):585-593
A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The
library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading
frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are
found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant
protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C
in the presence of 1 mM AgNO3. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional
conformation and function of the alginate lyase. 相似文献
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8.
Pui Yi Yung Catherine Burke Matt Lewis Suhelen Egan Staffan Kjelleberg Torsten Thomas 《Nucleic acids research》2009,37(21):e144
Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information. 相似文献
9.
Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes 总被引:1,自引:0,他引:1
Diego Javier Jiménez Fernando Dini Andreote Diego Chaves José Salvador Monta?a Cesar Osorio-Forero Howard Junca María Mercedes Zambrano Sandra Baena 《PloS one》2012,7(12)
10.
Huirong Li Yong Yu Wei Luo Yinxin Zeng Bo Chen 《Extremophiles : life under extreme conditions》2009,13(2):233-246
In order to assess bacterial diversity within four surface sediment samples (0–5 cm) collected from the Pacific Arctic Ocean,
16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from
four libraries and 13 distinct major lineages of Bacteria were identified (α, β, γ, δ and ε-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). α, γ, and δ-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The γ-Proteobacteria were the dominant
bacterial lineage, representing near or over 50% of the clones. Over 35% of γ-Proteobacteria clones of four clone library
were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition
was different among sampling sites, which potentially was related to geochemical differences. 相似文献
11.
Ono A Miyazaki R Sota M Ohtsubo Y Nagata Y Tsuda M 《Applied microbiology and biotechnology》2007,74(2):501-510
Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated
soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other
was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional
complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity
to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction
from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9
incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these
plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.
A.O. and R.M. contributed equally to this work. 相似文献
12.
Huang Y Lai X He X Cao L Zeng Z Zhang J Zhou S 《Marine biotechnology (New York, N.Y.)》2009,11(1):124-131
To access to the microbial genetic resources of deep-sea sediment by a culture-independent approach, the sediment DNA was
extracted and cloned into fosmid vector (pCC1FOS) generating a library of 39,600 clones with inserts of 24–45 kb. The clone
fss6 producing red-brown pigment was isolated and characterized. The pigment was identified as melanin according to its physico-chemical
characteristics. Subcloning and sequences analyses of fss6 demonstrated that one open reading frame (ORF2) was responsible
for the pigment production. The deduced protein from ORF2 revealed significant amino acid similarity to the 4-hydroxyphenylpyruvate
dioxygenase (HPPD) from deep-sea bacteria Idiomarina loihiensis. Further study demonstrated that the production of melanin was correlated with homogentistic acid (HGA). The p-hydroxyphenylpyruvate produced by the Escherichia coli host was converted to HGA through the oxidation reaction of introduced HPPD. The results demonstrate that expression of DNA
extracted directly from the environment might generate applicable microbial gene products. The construction and analysis of
the metagenomic library from deep-sea sediment contributed to our understanding for the reservoir of unexploited deep-sea
microorganisms. 相似文献
13.
The Roseobacter clade, belonging to the family Rhodobacteraceae of the class Alphaproteobacteria, is one of the major bacterial groups in marine environments. A remarkable wealth of diverse large plasmids has been detected
in members of this lineage. Here, we analysed the genome structure and extrachromosomal DNA content of four strains of the
roseobacter species Marinovum
algicola by pulsed-field gel electrophoresis. They were originally isolated from toxic dinoflagellates and possess multireplicon genomes
with sizes between 5.20 and 5.35 Mb. In addition to the single circular chromosomes (3.60–3.74 Mb), whose organisation seem
to be conserved, 9 to 12 extrachromosomal replicons have been detected for each strain. This number is unprecedented for roseobacters
and proposes a sophisticated regulation of replication and partitioning to ensure stable maintenance. The plasmid lengths
range from 7 to 477 kb and our analyses document a circular conformation for all but one of them, which might represent a
linear plasmid-like prophage. In striking contrast to other roseobacters, up to one-third of the genomic information (1.75 Mb)
is plasmid borne in Marinovum
algicola. The plasmid patterns of some strains are conspicuously different, indicating that recombination and conjugative gene transfer
are dominant mechanisms for microevolution within the Roseobacter clade. 相似文献
14.
Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences 总被引:1,自引:0,他引:1
The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated
with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal
samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences
showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences
were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C
Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and
functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further
confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the
microbial composition of avian fecal microbial communities. 相似文献
15.
Fiona Fouhy Lesley A. Ogilvie Brian V. Jones R. Paul Ross Anthony C. Ryan Eugene M. Dempsey Gerald F. Fitzgerald Catherine Stanton Paul D. Cotter 《PloS one》2014,9(9)
The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2–512x), increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth. 相似文献
16.
Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus,"
"T. brockianus,"
T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction
fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species
2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception
of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length.
Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14
strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were
found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging
to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical
taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains.
Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the
same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a
particular hot spring.
Received: 24 October 1994 / Accepted: 6 March 1995 相似文献
17.
Yongchang Ouyang Shikun Dai Lianwu Xie M. S. Ravi Kumar Wei Sun Huimin Sun Danling Tang Xiang Li 《Marine biotechnology (New York, N.Y.)》2010,12(3):318-325
Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated
with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds
are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular
weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial
DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms
concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery.
DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes,
and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library
demonstrated that the recovered HMW DNA is suitable for metagenomic library construction. 相似文献
18.
Choi JJ Alkharouf NW Schneider KT Matthews BF Frederick RD 《Functional & integrative genomics》2008,8(4):341-359
Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the
USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession
PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA
pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences
in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of
the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray
revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell
into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were
prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich
proteins, and xyloglucan endotransglycosylases.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
Changes in the bacterial populations of the highly alkaline saline soil of the former lake Texcoco (Mexico) following flooding 总被引:4,自引:0,他引:4
César Valenzuela-Encinas Isabel Neria-González Rocio J. Alcántara-Hernández Isabel Estrada-Alvarado Francisco Javier Zavala-Díaz de la Serna Luc Dendooven Rodolfo Marsch 《Extremophiles : life under extreme conditions》2009,13(4):609-621
Flooding an extreme alkaline-saline soil decreased alkalinity and salinity, which will change the bacterial populations. Bacterial
16S rDNA libraries were generated of three soils with different electrolytic conductivity (EC), i.e. soil with EC 1.7 dS m−1 and pH 7.80 (LOW soil), with EC 56 dS m−1 and pH 10.11 (MEDIUM soil) and with EC 159 dS m−1 and pH 10.02 (HIGH soil), using universal bacterial oligonucleotide primers, and 463 clone 16S rDNA sequences were analyzed
phylogenetically. Library proportions and clone identification of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Bacteroidetes, Firmicutes and Cloroflexi showed that the bacterial communities were different. Species and genera of the Rhizobiales, Rhodobacterales and Xanthomonadales
orders of the α- and γ-subdivision of Proteobacteria were found at the three sites. Species and genera of the Rhodospirillales, Sphingobacteriales, Clostridiales, Oscillatoriales
and Caldilineales were found only in the HIGH soil, Sphingomonadales, Burkholderiales and Pseudomonadales in the MEDIUM soil,
Myxococcales in the LOW soil, and Actinomycetales in the MEDIUM and LOW soils. It was found that the largest diversity at
the order and species level was found in the MEDIUM soil as bacteria of both the HIGH and LOW soils were found in it. 相似文献