Tyrosine Recombinase Family Research Articles

Functional characterization of Microplitis demolitor bracovirus genes that encode nucleocapsid components.

Understanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of the Microplitis demolitor bracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding protein p6.9-1 are required for DNA processing and packaging into nucleocapsids.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolic pathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotine to nicotine blue, alpha-ketoglutarate and succinate. Various modules of these genes have been shown to be present in gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bp plasmid pZXY21 of Arthrobacter sp. ZXY2 (96 percent to 100 percent at the nucleotide level) permitted the identification of the limits of this DNA fragment. At the 5' end of the nic-genes are located the ORFs of two predicted integrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of a small transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C of Staphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolic transposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encoding transposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide the nic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes can be mobilized and spread by horizontal gene transfer to other soil bacteria.

Identification and Characterization of oriT and Two Mobilization Genes Required for Conjugative Transfer of Salmonella Genomic Island 1.

The integrative mobilizable elements of SGI1-family considerably contribute to the spread of resistance to critically important antibiotics among enteric bacteria. Even though many aspects of SGI1 mobilization by IncA and IncC plasmids have been explored, the basic transfer elements such as oriT and self-encoded mobilization proteins remain undiscovered. Here we describe the mobilization region of SGI1 that is well conserved throughout the family and carries the oriTSGI1 and two genes, mpsA and mpsB (originally annotated as S020 and S019, respectively) that are essential for the conjugative transfer of SGI1. OriTSGI1, which is located in the vicinity of the two mobilization genes proved to be a 125-bp GC-rich sequence with several important inverted repeat motifs. The mobilization proteins MpsA and MpsB are expressed from a bicistronic mRNA, although MpsB can be produced from its own mRNA as well. The protein structure predictions imply that MpsA belongs to the lambda tyrosine recombinase family, while MpsB resembles the N-terminal core DNA binding domains of these enzymes. The results suggest that MpsA may act as an atypical relaxase, which needs MpsB for SGI1 transfer. Although the helper plasmid-encoded relaxase proved not to be essential for SGI1 transfer, it appeared to be important to achieve the high transfer rate of the island observed with the IncA/IncC-SGI1 system.

The integrase of the Macrococcus caseolyticus resistance island mecD (McRImecD ) inserts DNA site-specifically into Staphylococcus and Bacillus chromosomes.

The methicillin resistance gene mecD has been recently identified on chromosomal islands in Macrococcus caseolyticus (McRImecD ). The 5' end of McRImecD carries an integrase (int) of the tyrosine recombinase family and two genes (intR and xis) encoding putative DNA-binding proteins. The islands are integrated site-specifically at the 3' end of the rpsI gene, a highly conserved locus in Gram-positive bacteria. Moreover, the rpsI gene of some Staphylococcus and Bacillus strains was found to be followed by a related integrase, raising the question of whether McRImecD could be transferred to these species. We used circular model elements carrying 5' end fragments of McRImecD -1 to demonstrate that the int enzyme and the attachment (att) site were sufficient to mediate site-specific DNA integration into the rpsI locus of Staphylococcus aureus, Staphylococcus pseudintermedius and Bacillus thuringiensis in vivo. Including xis in the model element stimulated both integrative and excisive recombination reactions and influenced the Int enzyme in att site selection. The intR gene functions as a negative regulator of int and xis. The int-xis genes of McRImecD -1 encode a site-specific recombination function that enables the acquisition of McRImecD in new hosts and the potential dissemination of broad-spectrum β-lactam resistance across genus barriers.

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT.

CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation.IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.

Four genes essential for recombination define GInts, a new type of mobile genomic island widespread in bacteria

Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3′ end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.

Novel methicillin resistance gene mecD in clinical Macrococcus caseolyticus strains from bovine and canine sources

Methicillin-resistant Macrococcus caseolyticus strains from bovine and canine origins were found to carry a novel mecD gene conferring resistance to all classes of β-lactams including anti-MRSA cephalosporins. Association of β-lactam resistance with mecD was demonstrated by gene expression in S. aureus and deletion of the mecD-containing island in M. caseolyticus. The mecD gene was located either on an 18,134-bp M. caseolyticus resistance island (McRImecD-1) or a 16,188-bp McRImecD-2. Both islands were integrated at the 3′ end of the rpsI gene, carried the mecD operon (mecD-mecR1m-mecIm), and genes for an integrase of the tyrosine recombinase family and a putative virulence-associated protein (virE). Apart from the mecD operon, that shared 66% overall nucleotide identity with the mecB operon, McRImecD islands were unrelated to any mecB-carrying elements or staphylococcal cassette chromosome mec. Only McRImecD-1 that is delimitated at both ends by direct repeats was capable of circular excision. The recombined excision pattern suggests site-specific activity of the integrase and allowed identification of a putative core attachment site. Detection of rpsI-associated integrases in Bacillus and S. aureus reveals a potential for broad-host range dissemination of the novel methicillin resistance gene mecD.

The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution

DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed. Previous work showed that a valine (V95) in a predicted alpha helix in the N-terminus of IntDOT is required for resolution of HJs to substrates and products. We have identified two additional hydrophobic residues in the helix (A92 and F99) that are involved in resolution of HJs. IntDOT proteins with substitutions at these residues form aberrant complexes in an electrophoretic mobility shift assay. We propose that these three residues participate in hydrophobic interactions that are involved in forming higher-order complexes and resolution of HJs.

Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions.

SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (Int(SSV2)). Int(SSV2) alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of Int(SSV2) (residues 81 to 328), which corresponded to the putative product of the 3'-end sequence of the Int(SSV2) gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length Int(SSV2) except for the slightly reduced recombination activity. Our results suggest that, unlike λ integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion. Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.

Replication and Maintenance of Linear Phage-Plasmid N15.

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

Cre Recombinase.

The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

Xer Site-Specific Recombination: Promoting Vertical and Horizontal Transmission of Genetic Information.

Two related tyrosine recombinases, XerC and XerD, are encoded in the genome of most bacteria where they serve to resolve dimers of circular chromosomes by the addition of a crossover at a specific site, dif. From a structural and biochemical point of view they belong to the Cre resolvase family of tyrosine recombinases. Correspondingly, they are exploited for the resolution of multimers of numerous plasmids. In addition, they are exploited by mobile DNA elements to integrate into the genome of their host. Exploitation of Xer is likely to be advantageous to mobile elements because the conservation of the Xer recombinases and of the sequence of their chromosomal target should permit a quite easy extension of their host range. However, it requires means to overcome the cellular mechanisms that normally restrict recombination to dif sites harbored by a chromosome dimer and, in the case of integrative mobile elements, to convert dedicated tyrosine resolvases into integrases.

Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination.

The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.

Characterization of IntA, a Bidirectional Site-Specific Recombinase Required for Conjugative Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.

Promoter Orientation of Prokaryotic Phase-variable Genes by PCR.

One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic Escherichia coli (ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.

TPV1, the first virus isolated from the hyperthermophilic genus Thermococcus

We describe a novel virus, TPV1 (Thermococcus prieurii virus 1), which was discovered in a hyperthermophilic euryarchaeote isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2700 m at the East Pacific Rise. TPV1 is the first virus isolated and characterized from the hyperthermophilic euryarchaeal genus Thermococcus. TPV1 particles have a lemon-shaped morphology (140 nm × 80 nm) similar to the structures previously reported for Fuselloviruses and for the unclassified virus-like particle PAV1 (Pyrococcus abyssi virus 1). The infection with TPV1 does not cause host lysis and viral replication can be induced by UV irradiation. TPV1 contains a double-stranded circular DNA of 21.5 kb, which is also present in high copy number in a free form in the host cell. The TPV1 genome encompasses 28 predicted genes; the protein sequences encoded in 16 of these genes show no significant similarity to proteins in public databases. Proteins predicted to be involved in genome replication were identified as well as transcriptional regulators. TPV1 encodes also a predicted integrase of the tyrosine recombinase family. The only two genes that are homologous between TPV1 and PAV1 are TPV1-22 and TPV1-23, which encode proteins containing a concanavalin A-like lectin/glucanase domain that might be involved in virus-host recognition.

Analysis of the Site-Specific Integration System of the Streptomyces aureofaciens Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

Characterization of the Streptococcus suis XerS recombinase and its unconventional cleavage of the difSL site

XerC and XerD are members of the tyrosine recombinase family and mediate site-specific recombination that contributes to the stability of circular chromosomes in bacteria by resolving plasmid multimers and chromosome dimers to monomers prior to cell division. Homologues of xerC/xerD genes have been found in many bacteria, and in the lactococci and streptococci, a single recombinase called XerS can perform the functions of XerC and XerD. The xerS gene of Streptococcus suis was cloned, overexpressed and purified as a maltose-binding protein (MBP) fusion. The purified MBP-XerS fusion showed specific DNA-binding activity to both halves of the dif site of S.suis, and covalent protein-DNA complexes were also detected with dif site suicide substrates. These substrates were also cleaved in a specific fashion by MBP-XerS, generating cleavage products separated by an 11-bp spacer region, unlike the traditional 6-8-bp spacer observed in most tyrosine recombinases. Furthermore, xerS mutants of S.suis showed significant growth and morphological changes.

Resolution of Holliday Junction Recombination Intermediates by Wild-Type and Mutant IntDOT Proteins

CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.