Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.     

Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA

The region of temperate bacteriophage T12 responsible for integration into the chromosome of Streptococcus pyogenes has been identified. The integrase gene ( int ) and the phage attachment site ( attP ) are found immediately upstream of the gene for speA , the latter of which is known to be responsible for the production of erythrogenic toxin A (also known as pyrogenic exotoxin A). The integrase gene has a coding capacity for a protein of 41 457 Da, and the C-terminus of the deduced protein is similar to other conserved C-terminal regions typical of phage integrases. Upstream of int is a second open reading frame, which is capable of encoding an acidic protein of 72 amino acids (8744 Da); the position of this region in relation to int suggests it to be the phage excisionase gene ( xis ). The arms flanking the integrated prophage ( attL and attR ) were identified, allowing determination of the sequences of the phage ( attP ) and bacterial ( attB ) attachment sites. A fragment containing the integrase gene and attP was cloned into a streptococcal suicide vector; when introduced into S. pyogenes by electrotransformation, this plasmid stably integrated into the bacterial chromosome at attB . The insertion site for the phage into the S. pyogenes chromosome was found to be in the anticodon loop of a putative type II gene for a serine tRNA. attP and attB share a region of identity that is 96 bp in length; this region of identity corresponds to the 3' end of the tRNA gene such that the coding sequence remains intact after integration of the prophage. The symmetry of the core region of att may set this region apart from previously described phage attachment sites (Campbell, 1992), and may play a role in the biology of this medically important bacteriophage.     

Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3' end of a tRNA(Ser) gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA(Ser) gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.     

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3′ end of a putative proline tRNA gene

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 by region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3′ end of a putative proline tRNA gene. This gene shows 79% similartiy to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.     

Construction,characterization, and use of two Listeria monocytogenes site-specific phage integration vectors

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of approximately 10(-4) per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA(Arg) gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.     

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.     

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.     

The vibrio pathogenicity island of epidemic Vibrio cholerae forms precise extrachromosomal circular excision products

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. Like many pathogenicity islands, the VPI has at its termini a phage-like integrase gene (int), a transposase-like gene (vpiT), and phage-like attachment (att) sites, and is inserted at a tRNA-like locus (ssrA). We report that the VPI precisely excises from the chromosome and that its left and right ends join to form an extrachromosomal circular excision product (pVPI). Two-stage nested PCR analysis and DNA sequencing confirmed the int-att-vpiT junction and that the core attP of pVPI is identical to the chromosomal VPI attR site. Excision was independent of toxR and toxT. Excision was independent of recA, suggesting that it is mediated by site-specific recombination. Interestingly, while excision was detected in int and vpiT mutants, excision was abolished in a double (int vpiT) mutant and was restored by plasmids containing genes for either recombinase. Excision results in deletion of A361 in the ssrA locus, which flanks the right junction of the VPI. Since A361 encodes U70 in the critical G. U base pair in the acceptor stem of the ssrA RNA that is the determinant for aminoacylation with alanine, this deletion might have deleterious effects on ssrA function. Also, vpiT may have undergone interchromosomal translocation or may represent an independent integration event, as it was found downstream of hutA in some isolates. Our results provide new insight into the molecular biology of the VPI, and we propose that the process of excision and circularization is important in the emergence, pathogenesis, and persistence of epidemic V. cholerae.     

Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies

Most classical integrases of prokaryotic genetic elements specify integration into tRNA or tmRNA genes. Sequences shared between element and host integration sites suggest that crossover can occur at any of three sublocations within a tRNA gene, two with flanking symmetry (anticodon-loop and T-loop tDNA) and the third at the asymmetric 3' end of the gene. Integrase phylogeny matches this classification: integrase subfamilies use exclusively either the symmetric sublocations or the asymmetric sublocation, although tRNA genes of several different aminoacylation identities may be used within any subfamily. These two familial sublocation preferences imply two modes by which new integration site usage evolves. The tmRNA gene has been adopted as an integration site in both modes, and its distinctive structure imposes some constraints on proposed evolutionary mechanisms.     

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.     

Full length L1 retroposons contain tRNA-like sequences near the 5' termini--hypothesis on the replication mechanism of retroposons

Retrotransposons replicate via a complex mechanism which depends on, among other things, the presence of long terminal repeats (LTRs) and a tRNA binding site just 3' of the 5' LTR. The LINES 1 (L1) family of sequences, which similar to retrotransposons in many other properties, represents a new class of retroposon which does not possess LTRs. However, we show here that the repetitive 5' motif associated with murine L1 elements contains a tRNA-like sequence in a location analogous to the position of the retro-transposon tRNA binding site. Although the repetition of such a 5' motif has only been found associated with murine L1 elements, we have found an analogous tRNA-like sequence near the 5' ends of the L1 elements from each of the other analyzed species for which the L1 family has been characterized, that is rat (L1Rr), human (L1Hs), drosophila (I element) and trypanosome (INGI). The conservation of this tRNA-like sequence near the 5' terminus of L1 elements from such diverse species suggests that it plays a functional role in the life of the L1 class of retroposon.     

Integrative Genetic Element That Reverses the Usual Target Gene Orientation

A genetic element integrating site specifically into a prokaryotic gene usually carries a copy of the 3' portion of that gene that restores the active gene even as the original is disrupted. A cryptic element in Mesorhizobium loti instead carries a copy of the 5' end of the tRNA gene into which it integrated. This has implications for the evolution of new integrase-site combinations.     

Int-B13, an Unusual Site-Specific Recombinase of the Bacteriophage P4 Integrase Family, Is Responsible for Chromosomal Insertion of the 105-Kilobase clc Element of Pseudomonas sp. Strain B13

Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages R73 and P4, the clc element integrates into the 3′ end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration. In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3′-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.     

ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL, encoded within ssrA, serves as the site of integration of a region of Salmonella-specific sequence; adjacent to the 5' end of ssrA is another region of Salmonella-specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella, serves as an attachment site for Salmonella-specific sequences, and is required for the growth of phage P22.     

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.     

Integration of satellite bacteriophage P4 in Escherichia coli. DNA sequences of the phage and host regions involved in site-specific recombination

We determined the DNA sequences of regions essential for bacteriophage P4 integration. A 20 base-pair core sequence in both phage (P4attP) and host (P4attB) attachment regions contains the recombination site. In P4attP this sequence is flanked by five repeated sequences. A 1.3 x 10(3) base open reading frame codes for P4 integrase. Two possible promoters are upstream from P4int. One would be recognized by Escherichia coli RNA polymerase and may be repressed by integrase protein. The second would be recognized by RNA polymerase modified after infection by a P4 helper phage, P2. The P4attB core sequence is the 3' end of a leucine tRNA gene. Downstream from this tRNA in E. coli K-12 is a region homologous to P4int that may be part of a cryptic prophage.     

The nucleotide sequence of the large ribosomal RNA gene and the adjacent tRNA genes from rat mitochondria.

We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria.     

Analysis of repetitive sequence elements containing tRNA-like sequences.

Several repetitive sequence elements from diverse species share extensive sequence homology with tRNA molecules. Analysis of the tRNA-like sequences within these elements suggest that they have originated from authentic tRNA sequences. Elements containing tRNA-like sequences can be divided into three distinct groups whose members share extensive sequence homology, have similar sequence organization and have unique species distribution. We suggest that these three groups represent independent examples of retroposon families that have originated from tRNAs.