IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.     

用基于"Neo/E"的技术构建重组质粒

根据重组工程原理,建立了一种用于构建重组质粒的“neo／E”(抗生素／单酶切位点)选择与反选择新方法。首先采用PCR方法扩增出线性打靶分子：然后进行两步体内同源重组,(1)neo／E基因敲入,重组子呈现neo抗性表型;(2)目的基因替换M／E基因。用限制酶E消化时,发生第二步重组的DNA分子不能被消化,能够转化大肠杆菌受体菌DH5α。应用该方法构建了重组质粒pGL3-Basic PC1900T。PCR及测序鉴定证明：外源片段重组率为20％,所建立的重组工程选择与反选择新技术为质粒构建提供了新的解决方案。     

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.     

Diversity of gene cassette promoters in class 1 integrons from wastewater environments

The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.     

Increased levels of sigJ mRNA in late stationary phase cultures of Mycobacterium tuberculosis detected by DNA array hybridisation

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.     

Structural organization and mechanisms of mobility of the gene cassette coding for resistance to antibiotics and bacterial virulence factors

The review is focussed on two types of gene cassettes which are significant in bacterial variability. The first type are cassettes with antibiotic resistance genes; these are the smallest mobile genetic elements including a gene (most commonly an antibiotic resistance gene) and a short sequence acting as a recombination site. Sometimes these cassettes contain genes not responsible for antibiotic resistance but their functions are not yet known. The second type contains large clusters of genes coding for bacterial virulence factors. They were termed "pathogenicity islands" due to their difference in the percentage of G-C pairs in comparison with bacterial chromosomes, in which they are contained. The structural organization and mechanisms of mobility of various types of gene cassettes are discussed.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in <Emphasis Type="Italic">Shigella</Emphasis>

Integrons are gene capture and expression systems that are characterized by the presence of an integrase gene. This encodes an integrase, a recombined site, and a promoter. They are able to capture gene cassettes from the environment and incorporate them using site-specific recombination. The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is significant. In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes. Multiple and complex expression regulation mechanisms involving mobile genetic elements in integrons have been developed in the evolution of Shigella strains. Knowledge of the epidemiology and molecular mechanisms of antimicrobial resistance in this important pathogen is essential for the implementation of intervention strategies. This review was conducted to introduce the structures and functions of integrons in Shigella species and mechanisms that control integron-mediated events linked to antibiotic resistance.     

Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.     

New integrative method to generate Bacillus subtilis recombinant strains free of selection markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P(lysA) promoter with that of the P(blaP) beta-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.