Site-specific insertion of three structural gene cassettes in transposon Tn7.

Transposon Tn7 has been known to carry genes for resistance to trimethoprim and spectinomycin. A poorly expressed streptothricin resistance gene, identical to the sat gene found in transposons Tn1825 and Tn1826, was localized between the two mentioned genes in Tn7. The surroundings of all three resistance genes indicated site-specific insertion of genetic cassettes.     

Simultaneous insertion of two expression cassettes into adenovirus vectors

We have designed AdenoQuick, a fast and versatile method to construct first-generation adenoviral vectors that contain one or two transgenes in the E1 and/or the E3 region. The method is based on the reconstitution of the entire genome of the desired recombinant virus in E. coli and the subsequent transfection of the DNA in a helper cell line. Since the construction of large adenoviral plasmids is generally difficult and therefore rebuffing for inexperienced researchers, we have optimized the cloning strategy by using bacterial positive-selection markers and a set of specific restriction enzymes that allow for directional cloning. The system is 99% efficient and allows one to insert simultaneously two expression cassettes into the E1 and E3 regions of the adenovirus genome.     

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)     

产ESBLs大肠埃希菌整合子及其相关基因盒的研究

目的检测产超广谱β-内酰胺酶（ESBLs）大肠埃希菌中整合子的整合酶及插入的相关基因盒情况,分析整合子对细菌耐药性的影响。方法采用K-B琼脂扩散法对45株临床分离的产ESBLs大肠埃希菌进行药敏试验;应用PCR法检测45株产ESBLs大肠埃希菌Ⅰ类、Ⅱ类和Ⅲ类整合子;对Ⅰ类整合子阳性菌进行整合子相关基因盒检测。结果45株菌中有27株（60．0％）含有Ⅰ类整合子,没有检测到Ⅱ类和Ⅲ类整合子阳性菌。在Ⅰ类整合子阳性菌中,有23株携带Ⅰ类整合子相关基因盒（85．2％）,5种不同的基因盒图谱,片段大小在600～2322bp,分离自同一科室的部分菌株携带大小相同的基因盒;Ⅰ类整合子阳性菌株的耐药率高于整合子阴性的菌株。结论Ⅰ类整合子及整合子相关基因盒在产ESBLs大肠埃希菌株中分布广泛,整合子在细菌耐药中发挥作用。     

Evidence for dynamic exchange of qac gene cassettes between class 1 integrons and other integrons in freshwater biofilms

Class 1 integrons carried by pathogens have acquired over 100 different gene cassettes encoding resistance to antimicrobial compounds, helping to generate a crisis in the management of infectious disease. It is presumed that these cassettes originated from environmental bacteria, but exchange of gene cassettes has surprisingly never been demonstrated outside laboratory or clinical contexts. We aimed to identify a natural environment where such exchanges might occur, and determine the phylogenetic range of participating integrons. Here we examine freshwater biofilms and show that families of cassettes conferring resistance to quaternary ammonium compounds ( qac ) are found on class 1 integrons identical to those from clinical contexts, on sequence variants of class 1 integrons only known from natural environments, and on other diverse classes of integrons only known from the chromosomes of soil and freshwater Proteobacteria . We conclude that gene cassettes might be readily shared between different integron classes found in environmental, commensal and pathogenic bacteria. This suggests that class 1 integrons in pathogens have access to a vast pool of gene cassettes, any of which could confer a phenotype of clinical relevance. Exploration of this resource might allow identification of resistance or virulence genes before they become part of multi-drug-resistant human pathogens.     

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.     

Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter.

On the basis of a previous finding that the 7.8-kb HindIII-O fragment of the human cytomegalovirus strain Towne genome is nonessential for viral replication, we constructed a vector, pKM, that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using this vector, we constructed Towne-strain-derived recombinant virus in which a chimeric lacZ gene fused to the simian virus 40 promoter and a poly(A) signal were inserted in place of the O fragment. Two types of recombinants were obtained which carried the chimeric gene in opposite directions, beta-Galactosidase (beta Gal) was produced throughout the infection cycle in human embryonic lung cells infected with these recombinants, and the rate of its synthesis in the early stages of infection was comparable to that of synthesis of a 65-kDa viral glycoprotein, one of the abundantly produced viral proteins. The chimeric lacZ gene introduced was stable and no lacZ- revertants have been observed so far.     

Emergence of dhfrXVb and blaCARB-4 gene cassettes in class 1 integrons from clinical Pseudomonas aeruginosa isolated in Amazon region

Integrons play a role in horizontal acquisition and expression of genes, as well as gene reservoir, contributing for the resistance phenotype, particularly relevant to bacteria of clinical importance. We aimed to determine the composition and the organization of the class 1 integron variable region present in Pseudomonas aeruginosa clinical isolates from Brazil. Strains carrying class 1 integrons were resistant to the majority of antibiotics tested, except to imipenem and ceftazidime. Sequence analysis of the integron variable region revealed the presence of the blaCARB-4 gene into two distinct cassette arrays: aacA4-dhfrXVb-blaCARB-4 and aadB-aacA4-blaCARB-4. dhfrXVb gene cassette, which is rare in Brazil and in P. aeruginosa species, was found in one isolate. PFGE analysis showed the spread of blaCARB-4 among P. aeruginosa clones. The occurrence of blaCARB-4 and dhfrXVb in Brazil may contribute for developing resistance to clinically important antibiotics, and shows a diversified scenarium of these elements occurring in Amazon clinical settings, where no study about integron dynamics was performed to date.     

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.     

Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China

In this study,326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes.Minimum inhibitory concentration(MIC) testing indicated that the antimicrobial resistance of E.coli has increased since the 1970s.The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials,and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.     

Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes

Site-specific insertion of modified amino acids in proteins expressed in living cells is an emerging field holding great promise for elucidating protein structure-function relationships, expression levels, localization, and activation states in a complex milieu. To evaluate the efficiency of amino acids modified to carry either a nitroxide spin probe or a fluorescence probe, we have developed a screen using the levels of functional luciferase protein expressed in Xenopus oocytes. Natural and modified amino acids were targeted to position 14 in firefly luciferase using an amber mutation or introducing the four-codon nucleotide GGGU. Using the amber stop codon, the incorporation efficiencies of injected tRNA charged with the native phenylalanine residue, a fluorescent NBD-alanine, or nitroxide-labeled cysteine and tyrosine amino acids ranged from 1% to 18%. While the NBD-amino acid derivative gave higher incorporation levels, the EPR signals from the spin-labeled amino acids allow for the direct assessment of aminoacylation extent and stability. Applying the four-base codon for the first time in Xenopus oocytes, we found the incorporation efficiencies were significantly lowered compared to results using the three-base amber codon. The studies presented here provide quantitative assessment of protein expression levels when using nonsense suppression to site-specifically label proteins with spectroscopic probes in oocytes. Finally, the effect of a 77-base RNA aptamer known to inhibit the eucaryotic release factor of protein synthesis was tested for its influence on nonsense incorporation in Xenopus oocytes. The combination of A34 and charged suppressor tRNA produced a 3-fold increase in the expressed TAG(14)-luciferase level, compared to the use of charged suppressor tRNA alone.     

外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

Marine integrons containing novel integrase genes,attachment sites,attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome.     

Mobile gene cassettes and DNA integration elements

Integrons are unique natural systems for capturing and spreading the antibiotic resistance genes among Gram-negative bacteria. Gene transfer into small genomes and into plasmids is via site-specific recombination. Integrons act as receptors of antibiotic resistance cassettes. There are known more than 50 cassettes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptomycin, and other antibiotics. The structure of integrons and of gene cassettes are described and the mechanisms of capture, mobilization, and expression of cassettes considered.