A site-specific, conservative recombination system carried by bacteriophage P1. Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment.

The bacteriophage P1 genome carries an invertible C segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats. With insertion and deletion mutants of P1 derivatives the site-specific recombinase gene cin for C inversion) has been mapped adjacent to the C segment and the cix sites (for C inversion cross-over) have been located at the outside ends of the inverted repeats. Inversion of the C segment functions as a biological switch and controls expression of the gene(s) responsible for phage infectivity carried on the C segment. The cin gene product can promote recombination between a 'quasi- cix ' site on plasmid pBR322 and a cix site on P1 DNA. The junctions formed on the resulting co-integrate can also serve as cix sites. This observation implies a potential evolutionary process to bring genes under the control of a biological switch acting by DNA inversion.     

Site-specific recombinase genes in three Shigella subgroups and nucleotide sequences of a pinB gene and an invertible B segment from Shigella boydii.

Inversional switching systems in procaryotes are composed of an invertible DNA segment and a site-specific recombinase gene adjacent to or contained in the segment. Four related but functionally distinct systems have previously been characterized in detail: the Salmonella typhimurium H segment-hin gene (H-hin), phage Mu G-gin, phage P1 C-cin, and Escherichia coli e14 P-pin. In this article we report the isolation and characterization of three new recombinase genes: pinB, pinD, and defective pinF from Shigella boydii, Shigella dysenteriae, and Shigella flexneri, respectively. The genes pinB and pinD were detected by the complementation of a hin mutation of Salmonella and were able to mediate inversion of the H, P, and C segments. pinB mediated H inversion as efficiently as the hin gene did and mediated C inversion with a frequency three orders of magnitude lower than that of the cin gene. pinD mediated inversion of H and P segments with frequencies ten times as high as those for the genes intrinsic to each segment and mediated C inversion with a frequency ten times lower than that for cin. Therefore, the pinB and pinD genes were inferred to be different from each other. The invertible B segment-pinB gene cloned from S. boydii is highly homologous to the G-gin in size, organization, and nucleotide sequence of open reading frames, but the 5' constant region outside the segment is quite different in size and predicted amino acid sequence. The B segment underwent inversion in the presence of hin, pin, or cin. The defective pinF gene is suggested to hae the same origin as P-pin on e14 by the restriction map of the fragment cloned from a Pin+ transductant that was obtained in transduction from S. flexneri to E. coli delta pin.     

A 140 base-pair DNA segment from the kanamycin resistance region of plasmid R1 acts as an origin of replication and promotes site-specific recombination

A 140 base-pair DNA segment situated just upstream of the kanamycin resistance gene of transposon Tn2350, a transposon carried by the plasmid R1, was found to act as an origin of replication and allow autonomous replication of a plasmid composed only of the segment and of the tetracycline resistance gene of pBR322. This segment also promotes site-specific recombination: when cloned in pBR322 it promotes multimer formation in a recA- strain. If two copies are cloned on the same plasmid they promote either deletion or inversion of the intervening region, depending on their orientation relative to each other. DNA gyrase seems to be involved in this process since the inversion rate, in a plasmid carrying sequences in opposite orientations, varies in different nalidixic acid-resistant strains (gyr A mutants) independently isolated.     

Sequence of the site-specific recombinase gene cin and of its substrates serving in the inversion of the C segment of bacteriophage P1.

Inversion of the 4.2-kb C segment flanked by 0.6-kb inverted repeats on the bacteriophage P1 genome is mediated by the P1-encoded site-specific cin recombinase. The cin gene lies adjacent to the C segment and the C inversion cross-over sites cixL and cixR are at the external ends of the inverted repeats. We have sequenced the DNA containing the cin gene and these cix sites. The cin structural gene consists of 561 nucleotides and terminates at the inverted repeat end where the cixL site is located. Only two nucleotides in the cixL region differ from those in the cixR and they are within the cin TAA stop codon. The cin promoter was localized by transposon mutagenesis within a 0.1-kb segment, which contains probable promoter sequences overlapping with a 'pseudo-cix' sequence cixPp. In a particular mutant, integration of an IS1-flanked transposon into the cin control region promoted weak expression of the cin gene. The cin and cix sequences show homology with corresponding, functionally related sequences for H inversion in Salmonella and with cross-over sites for G inversion in phage Mu. Based on a comparison of the DNA sequences and of the gene organizations, a possible evolutionary relationship between these three inversion systems and the possible significance of the cixPp sequence in the cin promoter are discussed.     

Site-specific recombination events mediated by the DNA invertase Cin of bacteriophage P1 during transformation

Abstract Bacteriophage P1 encodes the site-specific recombinase Cin which promotes inversion of the C segment, thus controlling the P1 host range. Cin can also mediate inefficient inversion between the normal crossover site cixL and a quasi-crossover site cixQ 1 in inverted orientation. Inversion between cixL and cixQ 1 occurs more frequently in a short period of time after transformation with a plasmid carrying the cin gene, cixL and cixQ 1 than in an established transformant of the plasmid. This is also the case for Cin-mediated deletion on a plasmid containing the cin gene and directly repeated cix sites.     

The Min DNA inversion enzyme of plasmid p15B of Escherichia coli 15T−: a new member of the Din family of site-specific recombinases

Plasmid p15B is a bacteriophage P1-related resident of Escherichia coli 15T-. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site-specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site-specific DNA inversion 300-fold.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.

Integrons constitute a novel family of DNA elements which evolved by site-specific integration of discrete units between two conserved segments. On the In4 integron of Tn1696, a precisely inserted gene cassette of 1,549 bp conferring nonenzymatic chloramphenicol resistance (cmlA) is present between the streptomycin-spectinomycin resistance (aadA2) gene cassette and the 3'-conserved segment of the integron. In this study, we present the nucleotide sequence of the cmlA gene cassette of Tn1696, show its similarity to bacterial efflux systems and other transport proteins, and present evidence for alterations that its expression exerts on bacterial membranes. The cmlA gene cassette apparently carries its own promoter(s), a situation that has not heretofore been observed in the integrons of multiresistance plasmids and transposons of gram-negative bacteria. One or more of these promoters were shown to be functionally active in expressing a cat marker gene from promoter-probe vectors. The putative CmlA polypeptide appears to provoke a reduction of the content of the major porins OmpA and OmpC.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.     

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.     

Participation of the hup gene product in site-specific DNA inversion in Escherichia coli

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that DNA inversion mediated by hin, gin, pin and rci but not by cin is blocked in a hupA hupB double mutant, although inversions in these systems occur in the hupA or hupB single mutant as efficiently as in the wild-type strain. These findings show that HU protein participates in site-specific DNA inversion in E. coli and that only one subunit, either HU-1 or HU-2, is sufficient for this inversion.     

A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.