New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements.

Integrative genetic elements (IGEs) are mobile multigene DNA units that integrate into and excise from host bacterial genomes. Each IGE usually targets a specific site within a conserved host gene, integrating in a manner that preserves target gene function. However, a small number of bacterial genes are known to be inactivated upon IGE integration and reactivated upon excision, regulating phenotypes of virulence, mutation rate, and terminal differentiation in multicellular bacteria. The list of regulated gene integrity (RGI) cases has been slow-growing because IGEs have been challenging to precisely and comprehensively locate in genomes. We present software (TIGER) that maps IGEs with unprecedented precision and without attB site bias. TIGER uses a comparative genomic, ping-pong BLAST approach, based on the principle that the IGE integration module (i.e. its int-attP region) is cohesive. The resultant IGEs from 2168 genomes, along with integrase phylogenetic analysis and gene inactivation tests, revealed 19 new cases of genes whose integrity is regulated by IGEs (including dut, eccCa1, gntT, hrpB, merA, ompN, prkA, tqsA, traG, yifB, yfaT and ynfE), as well as recovering previously known cases (in sigK, spsM, comK, mlrA and hlb genes). It also recovered known clades of site-promiscuous integrases and identified possible new ones.

A novel family of tyrosine integrases encoded by the temperate pleolipovirus SNJ2.

Genomes of halophilic archaea typically contain multiple loci of integrated mobile genetic elements (MGEs). Despite the abundance of these elements, however, mechanisms underlying their site-specific integration and excision have not been investigated. Here, we identified and characterized a novel recombination system encoded by the temperate pleolipovirus SNJ2, which infects haloarchaeon Natrinema sp. J7-1. SNJ2 genome is inserted into the tRNAMet gene and flanked by 14 bp direct repeats corresponding to attachment core sites. We showed that SNJ2 encodes an integrase (IntSNJ2) that excises the proviral genome from its host cell chromosome, but requires two small accessory proteins, Orf2 and Orf3, for integration. These proteins were co-transcribed with IntSNJ2 to form an operon. Homology searches showed that IntSNJ2-type integrases are widespread in haloarchaeal genomes and are associated with various integrated MGEs. Importantly, we confirmed that SNJ2-like recombination systems are encoded by haloarchaea from three different genera and are critical for integration and excision. Finally, phylogenetic analysis suggested that IntSNJ2-type recombinases belong to a novel family of archaeal integrases distinct from previously characterized recombinases, including those from the archaeal SSV- and pNOB8-type families.

Virulence and resistance on various pathogens mediated by mobile genetic integrons via high flux assays.

BACKGROUND: Recognized as a resistance mechanism responsible for the emergence and prevalence of antimicrobial resistance, integron is widely distributed and spread among clinical microorganisms and play a key role in the dissemination of such antimicrobial resistance, which may eventually contribute to the unleashing of "Super Bugs" In this study, detection assays based on loop-mediated isothermal amplification (LAMP) methodologies targeting on class 1 to class 3 integrase genes was developed and evaluated. METHODS: LAMP methodology was employed to develop novel detection assays on class 1, 2 and 3 integrons. Firstly, this protocol was specifically designed to detect such integrons by targeting integrase genes intI1, intI2 and intI3. Development, evaluation and optimization of such LAMP assays was studied, including the reaction temperature, volumn, time, sensitivity and specificity of both primers and targets. A total of 1082 strains, including 397 integron positive and 685 integron negative microorganisms, were included for the application verification of the established LAMP assays. RESULTS: The indispensability of each primer was confirmed, and the optimal amplification was obtained under 63 °C for 45 min, with 25 µl reaction found to be the most cost-efficient volume. As application was concerned, all of the 397 integron-positive isolates yielded positive amplicons and other 685 integron-negative bacteria were negative for the integron-LAMP assays, revealing totaling 100% detection rate and specificity. CONCLUSIONS: The established integron-LAMP assays was demonstrated to be a valid and rapid detection method for integrons screening, which may aid in both the laboratory and clinical integron screening for microorganisms.

Integron diversity in bacterial communities of freshwater sediments at different contamination levels.

Integrons, genetic elements known to be involved in the adaptation of pathogenic bacteria, were first discovered in the clinical setting. However, they are ancient structures found in various environments. When clinical integrons have a low diversity, with three integrases and gene cassettes essentially encoding antibiotic resistance, in natural environments, integrons show a greater diversity, of both gene cassettes and integrases. Although a large number of gene cassettes from environmental samples have been identified, integrase diversity remains poorly documented, and has not yet been investigated in freshwater environments. The work presented here explores environmental integrons in sediments from a freshwater environment, with emphasis on integrases. Integron diversity in bacterial communities was analyzed at sampling stations with different contamination levels and contaminant types. A total of 684 integrase sequences were obtained and grouped into 322 previously undescribed integron classes, revealing a diversity wider than that previously expected in non-clinical environments. The bacterial community structures did not fully explain the integron diversity suggesting that integrase diversity could be influenced by contamination level, and that contaminant type could influence gene cassette diversity. These results provide further arguments for the involvement of integrons in the adaptation of bacterial communities in response to contaminants in natural environments.

A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene.

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5' end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature.

Phylogenetic analysis of Maverick/Polinton giant transposons across organisms.

Polintons are a recently discovered group of large transposable elements (<40Kb in size) encoding up to 10 different proteins. The increasing number of genome sequencing projects has led to the discovery of these elements in genomes of protists, fungi, and animals, but not in plants. The RepBase database of eukaryotic repetitive elements currently contains consensus sequences and information of 70 Polinton elements from 28 organisms. Previous phylogenetic analyses have shown the relationship of Polintons to linear plasmids, bacteriophages, and retroviruses. However, a comprehensive phylogenetic analysis of all known Polintons has been lacking. We retrieved the Polinton consensus sequences from the most recent version of RepBase, and compiled amino acid sequences for the two most common Polinton-specific genes, the DNA polymerase-B and retroviral-like integrase. Open reading frame predictions and homology comparisons revealed partial or full sequences for 54 polymerases and 55 Polinton integrases. Multiple sequence alignments portrayed conservation in several functional motifs of these proteins. Phylogenetic analyses based on Bayesian inference using single- and combined-gene datasets revealed seven distinct lineages of Polintons that broadly follow the tree of life. Two of the seven lineages are found within the same species, indicating that ancient divergences have been retained to this day.

A comparative study of class 1 integrons in Acinetobacter baumannii.

Multidrug resistance (MDR) in Acinetobacter baumannii is increasingly reported and has become a significant public concern. The method responsible for the acquisition of resistance genes via integrons from the environment or intra-species in A. baumannii remains to be understood. This study was performed to investigate the transmission route of these integrons using a comparative analysis of published A. baumannii complete genomes. The phylogenetic analysis of A. baumannii type 1 integrases (IntI1) showed that the integrons could be transferred across the two evolutionary lineages, the international clone I (IC I) and clone II (IC II) strains. In addition, the integrons in A. baumannii strains were mainly responsible for the transfer of resistance genes for two types of long-term usage antibiotics and antiseptics, such as aminoglycosides, chloramphenicol and the quaternary-ammonium-compound family. The in silico comparative analysis of known integron integrases revealed that the intI genes were phylogenetically related among A. baumannii strains and some microorganisms living in a sediment community, implicating that the integrons of A. baumannii might have originated from those microorganisms belonging to the ß-preoteobacterial class in the sediment environment. The data suggest that the gain of class 1 integrons in A. baumannii strains may have started before the antibiotic era. This report shows that the origins of A. baumannii class 1 integrons may be the soil environment and that the resistance genes included in integrons are horizontally transferred across all the A. baumannii genomes, including IC I and IC II.

Phylogeny and organization of recombinase in trio (RIT) elements.

Recombinase in trio (RIT) elements are composed of three adjacent tyrosine based site-specific recombinases that commonly occur in bacterial genomes. In this study, we examine RIT elements found in the genomes of strains from 63 different genera across 7 phyla of Eubacteria and examine the specific organization of these elements, their phylogenetic and environmental distribution, and their potential for mobility. We have found that each recombinase in this RIT arrangement is associated with a distinct sub-family of the tyrosine recombinases, and that the order and orientation of these sub-families is consistently maintained. We have determined that the distribution of these elements suggests that they are an ancient feature of bacterial genomes, but identical copies found within individual strains indicates that they are capable of intragenomic mobility. The occurrence of identical elements on both the main chromosome and one or more plasmids within individual strains, coupled with the finding that in some cases related genera are carrying highly similar RIT elements indicates that horizontal transfer has in some cases proceeded through a plasmid intermediate.

GIN transposons: genetic elements linking retrotransposons and genes.

In a previous work, we characterized a gene, called Gypsy Integrase 1 (GIN1), which encodes a protein very similar to the integrase domains present in Gypsy/Ty3 retrotransposons. I describe here a paralog of GIN1 and GIN2 and show that both genes are present in multiple vertebrates and that a likely homolog is found in urochordates. Surprisingly, phylogenetic and structural analyses support the counterintuitive idea that the GIN genes did not directly derive from retrotransposons but from a novel type of animal-specific DNA transposons, the GIN elements. These elements, described for the first time in this study, are characterized by containing a gene that encodes a protein that is also very similar to Gypsy/Ty3 integrases. It turns out that the sequences of the integrases encoded by GIN1 and GIN2 are more similar to those found in GIN elements than to those detected in retrotransposons. Moreover, several introns are in the same positions in the integrase-encoding genes of some GIN elements, GIN1 and GIN2. The simplest explanation for these results is that GIN elements appeared early in animal evolution by co-option of the integrase of a retrotransposon, they later expanded in multiple animal lineages, and, eventually, gave rise to the GIN genes. In summary, GIN transposons may be the "missing link" that explain how GIN genes evolved from retrotransposons. GIN1 and GIN2 may have contributed to control the expansion of GIN elements and Gypsy/Ty3 retrotransposons in chordates.

Worldwide prevalence of class 2 integrases outside the clinical setting is associated with human impact.

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (< or =98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised approximately 70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction ( approximately 40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (< or =98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.

A copia-like retrotransposon gene encoding gypsy-like integrase in a red alga, Porphyra yezoensis.

A genomic PCR fragment of 4581 bp, referred to as PyRE10G, was isolated from the red alga Porphyra yezoensis. PyRE10G contained a putative open reading frame encoding gag, protease, integrase, reverse transcriptase (RT), and RNase H, but one stop codon was present in the integrase region. Southern blot analysis revealed that PyRE10G exists as a single copy in the genome. From the order of gene arrangement of polyproteins, PyRE10G appears to be a copia-like retrotransposon. Amino acid sequences of PyRE10G RT and RNase H were closely related to those of copia-like retrotransposons. In contrast, the phylogenetic tree suggested that PyRE10G integrase stands within the gypsy elements and outside the copia group. PyRE10G is the first example of a chimeric composition of copia- and gypsy-like polyprotein genes in a single element, supporting the hypothesis that long terminal repeat-containing retrotransposons have evolved by fusion of ancestral RT/RNase H and other polyprotein genes.

Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents.

The lack of information about mobile DNA in deep-sea hydrothermal vents limits our understanding of the phylogenetic diversity of the mobile genome of bacteria in these environments. We used culture-independent techniques to explore the diversity of the integron/mobile gene cassette system in a variety of hydrothermal vent communities. Three samples, which included two different hydrothermal vent fluids and a mussel species that contained essentially monophyletic sulfur-oxidizing bacterial endosymbionts, were collected from Suiyo Seamount, Izu-Bonin, Japan, and Pika site, Mariana arc. First, using degenerate polymerase chain reaction (PCR) primers, we amplified integron integrase genes from metagenomic DNA from each sample. From vent fluids, we discovered 74 new integrase genes that were classified into 11 previously undescribed integron classes. One integrase gene was recorded in the mussel symbiont and was phylogenetically distant from those recovered from vent fluids. Second, using PCR primers targeting the gene cassette recombination site (59-be), we amplified and subsequently identified 60 diverse gene cassettes. In multicassette amplicons, a total of 13 59-be sites were identified. Most of these sites displayed features that were atypical of the features previously well conserved in this family. The Suiyo vent fluid was characterized by gene cassette open reading frames (ORFs) that had significant homologies with transferases, DNA-binding proteins and metal transporter proteins, while the majority of Pika vent fluid gene cassettes contained novel ORFs with no identifiable homologues in databases. The symbiont gene cassette ORFs were found to be matched with DNA repair proteins, methionine aminopeptidase, aminopeptidase N, O-sialoglycoprotein endopeptidase and glutamate synthase, which are proteins expected to play a role in animal/symbiont metabolism. The success of this study indicates that the integron/gene cassette system is common in deep-sea hydrothermal vents, an environment type well removed from anthropogenic disturbance.

Phage integrases: biology and applications.

Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.

Enhanced genome annotation using structural profiles in the program 3D-PSSM.

A method (three-dimensional position-specific scoring matrix, 3D-PSSM) to recognise remote protein sequence homologues is described. The method combines the power of multiple sequence profiles with knowledge of protein structure to provide enhanced recognition and thus functional assignment of newly sequenced genomes. The method uses structural alignments of homologous proteins of similar three-dimensional structure in the structural classification of proteins (SCOP) database to obtain a structural equivalence of residues. These equivalences are used to extend multiply aligned sequences obtained by standard sequence searches. The resulting large superfamily-based multiple alignment is converted into a PSSM. Combined with secondary structure matching and solvation potentials, 3D-PSSM can recognise structural and functional relationships beyond state-of-the-art sequence methods. In a cross-validated benchmark on 136 homologous relationships unambiguously undetectable by position-specific iterated basic local alignment search tool (PSI-Blast), 3D-PSSM can confidently assign 18 %. The method was applied to the remaining unassigned regions of the Mycoplasma genitalium genome and an additional 13 regions were assigned with 95 % confidence. 3D-PSSM is available to the community as a web server: http://www.bmm.icnet.uk/servers/3dpssm

Teaching a new dog old tricks?

The recently determined crystal structures of fragments of the human and vaccinia virus type IB topoisomerases reveal unexpected similarity with the lambda family of site-specific recombinases. The conservation of structure suggests a common mechanism, indicating that topoisomerase activity may be the consequence of uncoupling DNA strand cleavage/religation from synapsis.

Characterization of the lysogeny DNA module from the temperate Streptococcus thermophilus bacteriophage phi Sfi21.

Phage phi Sfi21, the only temperate Streptococcus thermophilus phage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I. Southern blot hybridizations demonstrated that the phi Sfi21-specific DNA was clustered in an approximately 6.6-kb-long region, the putative lysogeny module. Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C-terminal parts, respectively, of the cl-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressor ant with homology to phage P1. The molecular arrangement of the predicted orfs in phage phi Sfi21 was very similar to that of the lactococcal phage BK5-T. The transition from phi Sfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats. Sequence analysis identified a holin protein to the left of the lysogeny module. A site-specific deletion of 2.4 kb, which reproducibly transformed phi Sfi21 into a lytic phage, was localized in the lysogeny module. It was flanked at both sides by conspicuous DNA repeats. One repeat region reflected the DNA around the attP site, while the other reflected the putative genetic switch region between repressor and anti-repressor genes. S. thermophilus host Sfi1 transformed with a plasmid containing int and orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous phi Sfi21. Part of the int gene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance. We speculate on the possibility of a bipartite immunity system for the control of lysogeny in phi Sfi21.