海绵宏基因组文库构建及抗菌肽功能基因的初步筛选

宏基因组文库技术是利用未培养微生物基因资源的有效途径。成功构建澳大利亚厚皮海绵的Fosmid宏基因组文库,插入片段平均大小为36.8kb。利用建立的宏基因组文库,采用PCR技术初步筛选到具有编码抗菌肽的功能基因。这是我国首次尝试构建海绵宏基因组文库,对于今后开发利用海绵丰富的基因资源具有重要的意义。     

温室黄瓜根结线虫发生地土壤微生物宏基因组文库的构建及其一个杀线虫蛋白酶基因的筛选

【目的】本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。【方法】采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库,同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛。【结果】该文库库容31008个克隆,平均插入片段36.5kb,包含1.13Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选,通过功能初筛,筛选到1个含杀线虫蛋白酶基因的Fosmid克隆(pro12)。进一步构建和筛选出亚克隆(espro124a5),通过对基因结构进行了初步分析发现:espro124a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no.YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,是一种新型的丝氨酸蛋白酶,有其保守的催化三元组:Asp469、His541和Ser348。【结论】密度梯度离心法提取到的DNA纯度高、片段长,完全能满足构建宏基因组Fosmid文库的要求;同时,构建的宏基因组Fosmid文库库容大,有利于我们从中筛选其他的微生物基因资源。     

红树林土壤微生物宏基因组文库的构建及两种新颖的β-葡萄糖苷酶基因的鉴定

宏基因组技术是挖掘微生物新酶的重要途径。通过构建红树林土壤微生物Fosmid文库,共获得了约100 000个阳性克隆,该文库外源插入片段平均长度约为30 kb,文库容量达3 Gb。通过对文库中10 000个克隆进行功能筛选,获得了17个具有β-葡萄糖苷酶活性的克隆。对其中2个表达β-葡萄糖苷酶的克隆进行亚克隆,获得2个新颖的β-葡萄糖苷酶的基因,分别命名MhGH3和bgl66。生物信息学分析表明,MhGH3基因由1 992个碱基对组成,bgl66基因由2 025个碱基对组成。在氨基酸水平上,MhGH3和bgl66编码的蛋白质与GenBank数据库中已知β-葡萄糖苷酶的一致性分别为64%和55%。     

武夷菌素产生菌Fosmid文库的构建及文库探针的获得

以不吸水链霉菌武夷变种CK-15为材料,提取基因组DNA,经Sau 3A Ⅰ部分酶切后回收35-40 kb之间的片段,连接到pCC1FOS载体上,经过包装转染涂布后构建得到了CK-15基因组的Fosmid文库,文库的滴度为8.8×105CFU/mL.随机挑取16个阳性克隆,经EcoR Ⅰ和Hind Ⅲ双酶切电泳分析,样品插入片段平均长度大于35 kb,插入率为100%,符合构建文库的要求.根据多氧霉素、尼克霉素生物合成基因及大环内酯类聚酮合成酶基因(PKS)设计特异性引物,以基因组为模板进行PCR扩增,筛选特异性引物探针.结果用大环内酯类聚酮合成酶基因设计引物扩增出1 693 bp的片段经Blast比对其与大环内酯类抗生素生物合成基因相似性为95%以上.武夷菌素产生菌CK-15 Fosmid文库的构建及文库探针的获得,为武夷菌素生物合成基因的克隆奠定了基础.     

沼气池宏基因组文库中未培养微生物β-葡萄糖苷酶基因的克隆与鉴定

以间接提取法提取了沼气池样品的微生物宏基因组DNA,用柯斯质粒载体pWEB:TNC构建了一个含三万个克隆的沼气池宏基因组文库,对文库中的克隆随机分析表明,该文库的外源片段平均长度为40 kb,文库的总容量为1 .2×106kb。对其中的一个在七叶苷平板上显色的阳性克隆pGXN100进行进一步亚克隆、测序和序列分析。结果表明,pGXN100上有一个全长为1 863bp的ORF,编码621个氨基酸组成的蛋白质。将该基因命名为Unglu100。与产气克雷伯菌属的一个β-葡萄糖苷酶基因AN292在核苷酸和氨基酸水平上分别有76%和85%的同源性,利用SMART软件进行预测表明,Unglu100可能是PTS中β-葡萄糖苷酶特异性的转运蛋白组件。     

对虾养殖水环境宏基因组Fosmid文库的构建

采用包埋法提取大片段基因组DNA,通过低熔点琼脂糖酶法回收40 kb左右的DNA,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EP1300-T1R,构建对虾养殖水环境Fosmid文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存8 000个克隆,包括了大概8×108个微生物细胞.用几丁质酶筛选平板对文库进行初步筛选,筛选到4个阳性克隆子.本试验构建Fosmid文库,初步获取了对虾养殖生态系统的微生物遗传信息,对于从虾池原位环境鉴定并筛选具有潜在益生作用的功能微生物意义重大.     

新疆细毛羊2倍基因组BAC文库PCR筛选系统的构建与使用

本研究所构建的BAC文库覆盖了8倍新疆细毛羊的基因组,平均插入片段的大小为133kb,同时文库92．5%的克隆插入片段大于100kb,而且有部分克隆甚至大于300kb,假定绵羊的基因组含有3×10~6kb,根据文库的平均插入片段大小为133kb,从文库筛选到目的片段的概率为98．208%。为了验证文库有较好的覆盖率,构建了2倍基因组文库PCR筛选系统,并对位于新疆细毛羊20号染色体MHC基因邻近区段的DMB_EX2、MCMA36、CP73和BM1258 4个分子标记进行了筛选,得到的平均阳性克隆数为1．5个,从筛选结果来看,这与文库插入片段估计的8倍基因组覆盖率相当接近并且没有偏向,这使得本文库成为研究绵羊的功能基因、位置克隆和完善基因组物理图谱的极为有用的资源。     

利用高效的大片段DNA回收方法改良Fosmid文库的构建

外源DNA插入片段为40 kb左右的Fosmid文库在基因组学研究中有广泛的应用,但长期以来,40 kb外源片段的分离与纯化依赖于传统的切胶并电洗脱至透析袋的方法,难以得到足够量的DNA片段,极大降低Fosmid文库构建的成功率。通过改进全自动核酸/蛋白质回收系统SageELF的操作流程,建立一种简单、便捷、高效地回收40 kb左右DNA片段的方法,并用其成功地构建高质量的Fosmid文库。从文库中随机挑选的25个单克隆,经过测序及酶切分析,发现该文库中插入的DNA片段大小为37.9±5.2 kb。以上结果表明,利用改良的操作方法回收40 kb基因组DNA,操作简捷、高效,片段大小精准;另外,用该DNA片段构建的Fosmid文库,插入片段比较集中,有利于后续的基因组学分析。     

新疆军垦型细毛羊基因组BAC文库的构建

选用新疆军垦型细毛羊为材料,.构建了含有190 464个克隆的BAC文库,文库平均插入片段大小为133 kb,同时文库92.5%的克隆插入片段大于100 kb,而且有部分克隆甚至大于300 kb,这将满足大多数基因筛选的要求.假定绵羊的基因组含有3x106 kb,根据文库的平均插入片段大小,计算的文库基因组覆盖率为8倍.因此,从文库筛选到目的片段的概率为98.2%.由于该文库中插入的外源片段来自新疆军垦型细毛羊的基因组,这对于研究新疆军垦型细毛羊的特殊性状的基因与其他绵羊品种和物种之间的差异,及构建其全基因组物理图谱和基因图谱地完善是非常有利的.     

荷斯坦奶牛瘤胃微生物元基因组Fosmid文库的构建与分析

采用包埋法提取荷斯坦奶牛瘤胃微生物大片段总DNA,纯化后脉冲场电泳回收大小为36～48 kb,与pcc2FOS vector连接,转染至大肠埃希菌EPI 300宿主细胞,构建瘤胃微生物Fosmid基因组文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存30 000个克隆,空载体率小于2%,库容达1 050 Mb.     

Uncultured soil bacteria are a reservoir of new antibiotic resistance genes

Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.     

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.     

Several biological properties of E. coli SK cells]

Escherichia coli SK cells are not colicinogenic, but possess multiple resistance to antibiotics (tetracycline, kanamycin, penicillin, polymyxin, ampicillin). Numerous variations, sensitive to one or several of the antibiotics listed were obtained by cloning the initial culture. Experiments with acridine orange failed to eliminate completely the resistance of E. coli cells to all five of the antibiotics under study. Partial modifications in the spectrum of antibiotic resistance did not influence the host specificity system present in the cells SK. The capacity of restriction and the activity of the methylating enzymes in all the clones under study in the initial strain proved to be the same.     

A multiresistant clone of Shiga toxin-producing Escherichia coli O118:[H16] is spread in cattle and humans over different European countries

Multiresistant Shiga toxin-producing Escherichia coli (STEC) O118:H16 and O118 nonmotile strains (designated O118:[H16]) were detected by examination of 171 STEC isolates for their antimicrobial sensitivity. Of 48 STEC O118:[H16] strains, 98% were resistant to sulfonamide, 96% were resistant to streptomycin, 79% were resistant to kanamycin, 75% were resistant to tetracycline, 67% were resistant to ampicillin, 60% were resistant to chloramphenicol, 48% were resistant to trimethoprim, and 10% each were resistant to gentamicin and nalidixic acid. Nalidixic acid resistance and reduced susceptibility to ciprofloxacin were associated with the mutation gyrA(LEU-83). The STEC O118:[H16] strains were found to belong to a single genetic clone as investigated by multilocus enzyme electrophoresis and by multilocus sequence analysis of E. coli housekeeping genes. The STEC O118:[H16] strains originated from humans and cattle and were isolated in seven different countries of Europe between 1986 and 1999. Strains showing multiresistance to up to eight different antimicrobials predominated among the more recent STEC O118:[H16] strains. The genes in parentheses were associated with resistance to kanamycin (aphA1-Ia), chloramphenicol (catA1), tetracycline [tet(A)], and ampicillin (bla(TEM-1)). Class 1 integrons containing sulI (sulfonamide resistance), aadA1a (streptomycin resistance), or dfrA1 (trimethoprim resistance)-aadA1a gene cassettes were detected in 28 strains. The bla(TEM-1b) gene was present in 18 of 21 strains that were examined by nucleotide sequencing. Class 1 integrons and bla(TEM) genes were localized on plasmids and/or on the chromosome in different STEC O118:[H16] strains. Hybridization of XbaI-digested chromosomal DNA separated by pulsed-field gel electrophoresis revealed that bla(TEM) genes were integrated at different positions in the chromosome of STEC O118:[H16] strains that could have occurred by Tn2 insertion. Our data suggest that strains belonging to the STEC O118:[H16] clonal group have a characteristic propensity for acquisition and maintenance of resistance determinants, thus contrasting to STEC belonging to other serotypes.     

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Aquaculture Can Promote the Presence and Spread of Antibiotic-Resistant Enterococci in Marine Sediments

Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside) collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M), tet(L), tet(O)] and macrolide [erm(A), erm(B) and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M) and tet(L) in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin) highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O), erm(B) and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an underestimated health risk and deserves further investigation.     

Detection of Salmonella spp. in retail raw food samples from Vietnam and characterization of their antibiotic resistance

A study was conducted to examine the levels of Salmonella spp. contamination in raw food samples, including chicken, beef, pork, and shellfish, from Vietnam and to determine their antibiotic resistance characteristics. A total of 180 samples were collected and examined for the presence of Salmonella spp., yielding 91 Salmonella isolates. Sixty-one percent of meat and 18% of shellfish samples were contaminated with Salmonella spp. Susceptibility of all isolates to a variety of antimicrobial agents was tested, and resistance to tetracycline, ampicillin/amoxicillin, nalidixic acid, sulfafurazole, and streptomycin was found in 40.7%, 22.0%, 18.7%, 16.5%, and 14.3% of the isolates, respectively. Resistance to enrofloxacin, trimethoprim, chloramphenicol, kanamycin, and gentamicin was also detected (8.8 to 2.2%). About half (50.5%) of the isolates were resistant to at least one antibiotic, and multiresistant Salmonella isolates, resistant to at least three different classes of antibiotics, were isolated from all food types. One isolate from chicken (serovar Albany) contained a variant of the Salmonella genomic island 1 antibiotic resistance gene cluster. The results show that antibiotic resistance in Salmonella spp. in raw food samples from Vietnam is significant.