Mobile DNA Elements Research Articles

Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

High Prevalence of Multidrug Resistance and Random Distribution of Mobile Genetic Elements Among Uropathogenic Escherichia coli (UPEC) of the Four Major Phylogenetic Groups

One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

Broken Symmetry in Homing Endonucleases

Homing DNA endonucleases are highly site-specific enzymes that initiate the transfer of mobile DNA elements. In this issue of Structure , Spiegel et al. report the structure of the I-CeuI homing enzyme and describe how a symmetric homodimeric enzyme acquired specificity for an asymmetric substrate.

Structural basis for broad DNA-specificity in integron recombination

Lateral DNA transfer--the movement of genetic traits between bacteria--has a profound impact on genomic evolution and speciation. The efficiency with which bacteria incorporate genetic information reflects their capacity to adapt to changing environmental conditions. Integron integrases are proteins that mediate site-specific DNA recombination between a proximal primary site (attI) and a secondary target site (attC) found within mobile gene cassettes encoding resistance or virulence factors. The lack of sequence conservation among attC sites has led to the hypothesis that a sequence-independent structural recognition determinant must exist within attC. Here we report the crystal structure of an integron integrase bound to an attC substrate. The structure shows that DNA target site recognition and high-order synaptic assembly are not dependent on canonical DNA but on the position of two flipped-out bases that interact in cis and in trans with the integrase. These extrahelical bases, one of which is required for recombination in vivo, originate from folding of the bottom strand of attC owing to its imperfect internal dyad symmetry. The mechanism reported here supports a new paradigm for how sequence-degenerate single-stranded genetic material is recognized and exchanged between bacteria.

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104.

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacE delta 1 genes characteristic of integrons. The first integron encoded the ant (3")-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 beta-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.

DNA methyltransferase-3a interacts with p53 and represses p53-mediated gene expression

Genome stability maintenance is regulated by both genetic and epigenetic mechanisms. DNA methylation is the predominant epigenetic mechanism in regulation of gene expression and in suppression of mobile DNA elements from random integration in the genome. The importance of DNA methylation in tumorigenesis has been demonstrated in cancer cells which harbor global genomic DNA hypomethylation and regional hypermethylation at CpG islands of tumor suppressor genes. DNA methylation is mediated by a class of DNA methyltransferases (Dnmts) involved in de novo methylation of genomic DNA and in the maintenance of DNA methylation patterns during replication. Global genomic DNA demethylation induced by 5-Aza-deoxycytidine activates the p53 signaling pathway and induces apoptosis, suggesting that DNA methylation mediated by Dnmts is associated with p53 signaling in maintaining genome stability. In this report, we show that Dnmt3a interacts with p53 directly and represses p53-mediated transactivation of the p21 gene. It was found that trans-repression by Dnmt3a does not require the methyltransferase activity implying that transcriptional repression does not involve promoter silencing through DNA methylation by Dnmt3a. Finally, the activity of Dnmt3a in vivo was demonstrated when this enzyme was overexpressed in a breast cell line in which Dnmt3a repressed p21 upregulation following DNA damage. The results presented in this study provide new understanding of tumor promotion as mediated by Dnmt3a through its interaction with p53, and suppression of the p53-mediated transcription of tumor suppressor genes. Given that the expression of Dnmts is increased in certain cancers, it is likely that increased Dnmts could block the transactivation function of p53 following its induction by chemotherapeutic drugs resulting in chemoresistance. The use of a DNA methyltransferase inhibitor would therefore restore the p53 tumor suppression function and the utilization of such an inhibitor in combination with DNA damage agents might be an effective therapy for certain cancers.

HATpin, a family of MITE-like hAT mobile elements conserved in diverse plant species that forms highly stable secondary structures.

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species.

Risk factors associated with extended-spectrum β-lactamase-producing organisms at a tertiary care hospital

In 1995, beta-lactam inhibitor combinations replaced third-generation cephalosporins as empirical therapy in an effort to manage extended-spectrum beta-lactamase (ESBL) resistance. This study investigated the relationship between antibiotic usage and ESBL organisms from 1994 through 2002 using epidemiological and molecular analysis. A case-control study of 119 patients with ESBL organisms and 132 patients with non-ESBL organisms was conducted. Demographics, co-morbidities, device utilization and antibiotic use were analysed for all patients and infected patients only (cases = 75, controls = 83). Both exposure and degree of exposure (in grams) to antibiotics were included. A dot blot hybridization technique was used to identify genes in plasmid extracts from the ESBL organisms. Ventilator days OR 1.1 (1.06, 1.15) P < 0.001, adult respiratory distress syndrome (ARDS) OR 3.1 (1.0, 9.7) P = 0.05, prior aminoglycoside use OR 2.7 (1.2, 6.1) P = 0.02, prior third-generation cephalosporin use OR 7.2 (2.6, 20) P < 0.001, and prior trimethoprim/sulfamethoxazole use OR 8.8 (3.1, 26) P < 0.001 were significantly associated with ESBL organisms by multivariate analysis. All models were concordant with a significant association of ventilator days, third-generation cephalosporins and trimethoprim/sulfamethoxazole with ESBL organisms. beta-Lactamase inhibitor combinations were not associated with ESBL organisms. Hybridization of plasmid extracts demonstrated that 95% of the ESBL organisms carried intI1, a mobile DNA element with a sulphonamide-resistance (R) gene and a frequent carrier of other R factors. Genes for specific types of trimethoprim-R and aminoglycoside-R were present in 26% and 40% of the extracts, respectively. These data indicate that, besides patient risk factors and third-generation cephalosporins, other antibiotics may provide selective pressures in maintaining ESBL organisms due to multiple resistance genes on plasmids. beta-Lactamase inhibitor combinations appear to be an acceptable substitute to third-generation cephalosporins in strategies to control ESBL organisms.

Ecological implications of the emergence of non‐toxic subcultures from toxic Microcystis strains

Two toxic, microcystin-producing, Microcystis sp. strains KLL MG-K and KLL MB-K were isolated as single colonies on agar plates from Lake Kinneret, Israel. Two non-toxic subcultures, MG-J and MB-J spontaneously succeeded the toxic ones under laboratory conditions. Southern analyses showed that MG-J and MB-J are lacking at least 34 kb of the mcy region, encoding the microcystin synthetase. Analyses of the 16S rRNA genes, the intergenic spacer region between cpcB and cpcA and the patterns of the polymerase chain reaction products of randomly amplified polymorphic DNA and highly iterated palindrome, and presence of mobile DNA elements did not allow unequivocal distinction between toxic and non-toxic subcultures. Laboratory and field experiments indicated an advantage of the toxic strain over its non-toxic successor. When grown separated by a membrane, which allowed passage of the media but not the cells, MG-K severely inhibited the growth of MG-J. Furthermore, when MG strains were placed in dialysis bags in Lake Kinneret during the season in which Microcystis is often observed, cells of MG-J lysed, whereas MG-K survived. Mechanisms whereby the non-toxic subcultures emerged and prevailed over the corresponding toxic ones under laboratory conditions, as well as a possible role of microcystin under natural conditions, are discussed.

Comparative Study of Class 1 Integron andVibrio choleraeSuperintegron Integrase Activities

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.

The ecology of the genome — mobile DNA elements and their hosts

The genomes of multicellular eukaryotes provide information that determines the phenotype. However, not all sequences in the genome are required for this purpose. Other sequences are often selfish in their actions and interact in complex ways. Here, an analogy is developed between the components of the genome, including mobile DNA elements, and an ecological community. Unlike ecological communities, however, the slow rates at which genomes change allow us to reconstruct patterns of interaction that stretch back tens or hundreds of millions of years.

Comparative Genomics of the T4-Like Escherichia coli Phage JS98: Implications for the Evolution of T4 Phages

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.

A truncated H‐NS‐like protein from enteropathogenic Escherichia coli acts as an H‐NS antagonist

The H-NS nucleoid-associated protein of Escherichia coli is the founder member of a widespread family of gene regulatory proteins which have a bipartite structure, consisting of an N-terminal coiled-coil oligomerization domain and a C-terminal DNA-binding domain. Here we characterize a family of naturally occurring truncated H-NS derivatives lacking the DNA-binding domain, which we term the H-NST family. H-NST proteins are found in large genomic islands in pathogenic E. coli strains, which are absent from the corresponding positions in the E. coli K-12 genome. Detailed analysis of the H-NST proteins from enteropathogenic E. coli (EPEC) and uropathogenic E. coli (UPEC) shows that the EPEC protein (H-NST(EPEC)) has a potent anti-H-NS function at the classical H-NS-repressed operon proU. This correlates with the ability of H-NST(EPEC) to co-purify with H-NS in vitro, and can be abolished by a mutation of leucine 30 to proline which is predicted to prevent the N-terminal region from forming a coiled-coil structure. In contrast, despite being 90% identical to H-NST(EPEC) at the protein level, the UPEC homologue (H-NST(UPEC)) has only a weak anti-H-NS activity, correlating with a much-reduced ability to interact with H-NS during column chromatography. A single amino acid difference at residue 16 appears to account for these different properties. The hnsT(EPEC) gene is transcribed monocistronically and expressed throughout the exponential growth phase in DMEM medium. Our data suggest that a truncated derivative of H-NS encoded by an ancestral mobile DNA element can interact with the endogenous H-NS regulatory network of a bacterial pathogen.

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages

The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (approximately 900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3 -exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.

Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

Genes without frontiers?

For bacteria, the primary genetic barrier against the genetic exchange of DNA that is not self-transmissible is dissimilarity in the bacterial DNA sequences concerned. Genetic exchange by homologous recombination is frequent among close bacterial relatives and recent experiments have shown that it can enable the uptake of closely linked nonhomologous foreign DNA. Artificial vectors are mosaics of mobile DNA elements from free-living bacterial isolates and so bear a residual similarity to their ubiquitous natural progenitors. This homology is tightly linked to the multitude of different DNA sequences that are inserted into synthetic vectors. Can homology between vector and bacterial DNA enable the uptake of these foreign DNA inserts? In this review we investigate pUC18 as an example of an artificial vector and consider whether its homology to broad host-range antibiotic resistance transposons and plasmid origins of replication could enable the uptake of insert DNA in the light of studies of homology-facilitated foreign DNA uptake. We also discuss the disposal of recombinant DNA, its persistence in the environment and whether homologies to pUC18 may exist in naturally competent bacteria. Most DNA that is inserted into the cloning site of artificial vectors would be of little use to a bacterium, but perhaps not all.

Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.

The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis

Two non-inducible, but apparently complete prophages were identified in the genome of the sequenced Lactobacillus johnsonii strain NCC 533. The 38- and 40-kb-long prophages Lj928 and Lj965 represent distinct lineages of Sfi11-like pac-site Siphoviridae unrelated at the DNA sequence level. The deduced structural proteins from Lj928 demonstrated aa sequence identity with Lactococcus lactis phage TP901-1, while Lj965 shared sequence links with Streptococcus thermophilus phage O1205. With the exception of tRNA genes, inserted between DNA replication and DNA packaging genes, the transcription of the prophage was restricted to the genome segments near both attachment sites. Transcribed genes unrelated to phage functions were inserted between the phage repressor and integrase genes; one group of genes shared sequence relatedness with a mobile DNA element in Staphylococcus aureus. A short, but highly transcribed region was located between the phage lysin and right attachment site; it lacked a protein-encoding function in one prophage.