多重耐药肺炎克雷伯菌获得性耐药相关基因和可移动遗传元件检测与指标聚类分析

目的 探讨一组多重耐药肺炎克雷伯菌(MDR-KPN)中获得性耐药相关基因和可移动遗传元件遗传标记的存在状况以及二者的相关性.方法 收集2008年8月至2010年5月浙江省杭州市和湖州市6所医院共47株MDR-KPN,采用聚合酶链反应(PCR)的方法分析74种获得性耐药基因和24种可移动遗传元件遗传标记,并用指标聚类分析(SPSS法)分析获得性耐药相关基因和可移动遗传元件遗传标记的相关性.结果 47株MDR-KPN共检出5种β-内酰胺类获得性耐药基因、6种氨基糖苷类获得性耐药基因、3种喹诺酮类获得性耐药基因、6种其他获得性耐药基因、1种整合子遗传标记、2种转座子遗传标记、4种插入序列遗传标记、2种接合性质粒遗传标记和1种噬菌体原标记;指标聚类分析(SPSS法)将上述阳性检出基因分成A、B两大簇.结论 指标聚类分析提示获得性耐药相关基因和可移动遗传元件密切相关;由Ⅰ类整合子( intI1)、插入序列(IS26、ISEcp1、ISKpn6)、耐药质粒(trbC)介导的TEM-1和KPC是本组菌株的特征.在肺炎克雷伯菌中做指标聚类分析为国内首次报道.     

Insertion sequences enrichment in extreme Red sea brine pool vent

Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world’s largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.

     

Straightening out the LINEs: LINE-1 orthologous loci

The L1Hs preTa subfamily of long interspersed elements (LINEs) originated after the divergence of human and chimpanzee and is therefore found only in the human genome. Thirty-three of the 254 L1Hs preTa elements are polymorphic for the absence/presence of the insertion, making them useful markers for studying human population genetics. The problem of homoplasy, however, can diminish the value of LINEs as phylogenetic and population genetic markers. We examined anomalous orthologous sites in a range of nonhuman primates. Only two cases of other mobile elements inserting near the preintegration sites of L1Hs preTa elements were observed: an AluY insertion in Chlorocebus and an L1PA8 insertion in Aotus. Sequence analysis showed that both elements were clearly distinguishable from their human counterparts. We conclude that L1 elements can continue to be regarded as essentially homoplasy-free genetic characters.     

Mobile elements and mammalian genome evolution

Mobile elements make up large portions of most eukaryotic genomes. They create genetic instability, not only through insertional mutation but also by contributing recombination substrates, both during and long after their insertion. The combination of whole-genome sequences and the development of innovative new assays to test the function of mobile elements have increased our understanding of how these elements mobilize and how their insertion impacts genome diversity and human disease.     

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.     

Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.     

The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Insertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.     

Following the LINEs: an analysis of primate genomic variation at human-specific LINE-1 insertion sites

The L1 Ta subfamily of long interspersed elements (LINEs) consists exclusively of human-specific L1 elements. Polymerase chain reaction-based screening in nonhuman primate genomes of the orthologous sites for 249 human L1 Ta elements resulted in the recovery of various types of sequence variants for approximately 12% of these loci. Sequence analysis was employed to capture the nature of the observed variation and to determine the levels of gene conversion and insertion site homoplasy associated with LINE elements. Half of the orthologous loci differed from the predicted sizes due to localized sequence variants that occurred as a result of common mutational processes in ancestral sequences, often including regions containing simple sequence repeats. Additional sequence variation included genomic deletions that occurred upon L1 insertion, as well as successive mobile element insertions that accumulated within a single locus over evolutionary time. Parallel independent mobile element insertions at orthologous loci in distinct species may introduce homoplasy into retroelement-based phylogenetic and population genetic data. We estimate the overall frequency of parallel independent insertion events at L1 insertion sites in seven different primate species to be very low (0.52%). In addition, no cases of insertion site homoplasy involved the integration of a second L1 element at any of the loci, but rather largely involved secondary insertions of Alu elements. No independent mobile element insertion events were found at orthologous loci in the human and chimpanzee genomes. Therefore, L1 insertion polymorphisms appear to be essentially homoplasy free characters well suited for the study of population genetics and phylogenetic relationships within closely related species.     

Mobile Element Insertions Causing Mutations in the Drosophila Suppressor of Sable Locus Occur in Dnase I Hypersensitive Subregions of 5''-Transcribed Nontranslated Sequences

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.     

Global mapping of transposon location

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.     

Isolation and characterization of functional insertion sequences of rhizobia

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.     

Co‐evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella

Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF‐Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. We measured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but for insertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...     

插入序列共同区元件：细菌中新出现的一种基因捕获系统

摘要：插入序列共同区（Insertion sequence common region,ISCR）元件是一类在结构和功能上与IS91家族相似的特殊插入序列,特点是缺少了末端反向重复序列（Inverted repeats, IRs）,在插入位点不产生直接重复序列,并通过滚环式（Rolling circle, RC）进行转座。ISCR元件作为一种新的基因捕获系统,它可以移动邻近的任何DNA序列,为耐药基因在不同种属细菌间水平传播提供了高效的媒介。世界各地多种革兰氏阴性病原菌中已发现有19种ISCR元件,大部分ISCR元件同时携带了多种耐药基因,提示ISCR有可能会造成细菌多重耐药性的快速传播。本文就ISCR结构特征、类型、移动方式、起源及进化的研究进展进行了综述。     

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.     

CRISPR相关转座元件的研究及应用进展

CRISPR-Cas的基因编辑能力引发了人们对该系统的研究热潮。除了实现基因的敲除和插入,CRISPR-Cas系统还可以被应用于基因簇重组、单碱基编辑和基因转录调控,推动了生物工程领域的发展。然而,有限的同源重组效率使CRISPR-Cas系统的应用受到了一定的限制。与CRISPR-Cas系统相比,移动遗传元件(mobile genetic elements,MGE)在转座酶的调控下,不需要依赖同源重组即可将指定DNA片段定向插入到细胞染色体中。近几年,人们发现了具有转座机制的CRISPR相关的转座元件,它可以介导DNA靶向整合,同时其出色的重编程能力为该领域的研究带来了新的发展。本文主要介绍近年来CRISPR-Cas系统相关转座元件的研究方向和应用进展,以及人工融合的dCas9-transposase系统的应用策略。文中还提出了CRISPR相关转座元件未来的应用前景和潜在挑战,为基因编辑工具的发展方向提供了参考意见。     

Ecological and molecular maintenance strategies of mobile genetic elements

This review considers the influence of selection pressure, fitness and population structures on the evolution of mobile genetic elements (including plasmids, phage, pathogenicity islands, transposons and insertion sequences) that constitute the horizontal gene pool of bacteria. These are considered at different scales using examples from in vitro evolutionary studies of Escherichia coli and associated bacteriophage, detailed molecular analyses of the broad host-range IncP-1 plasmids, population surveys of pseudomonad plasmids and genomic comparisons of members of the Rhizobiaceae. All biological systems show genetic redundancy (the existence of allelic variation) at some population level, i.e. within a cell, a clone, population or community. We consider the level(s) at which redundancy is expressed and how this will affect and has influenced the evolution of mobile genetic elements.