Accessory genes in the darA operon of bacteriophage P1 affect antirestriction function, generalized transduction, head morphogenesis, and host cell lysis.

Bacteriophage P1 mutants with the 8.86-kb region between the invertible C-segment and the residential IS1 element deleted from their genome are still able to grow vegetatively and to lysogenize stably, but they show several phenotypic changes. These include the formation of minute plaques due to delayed cell lysis, the abundant production of small-headed particles, a lack of specific internal head proteins, sensitivity to type I host restriction systems, and altered properties to mediate generalized transduction. In the wild-type P1 genome, the accessory genes encoding the functions responsible for these characters are localized in the darA operon that is transcribed late during phage production. We determined the relevant DNA sequence that is located between the C-segment and the IS1 element and contains the cin gene for C-inversion and the accessory genes in the darA operon. The darA operon carries eight open reading frames that could encode polypeptides containing >100 amino acids. Genetic studies indicate that some of these open reading frames, in particular those residing in the 5' part of the darA operon, are responsible for the phenotypic traits identified. The study may contribute to a better comprehension of phage morphogenesis, of the mobilization of host DNA into phage particles mediating generalized transduction, of the defense against type I restriction systems, and of the control of host lysis.

Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome.

The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1. Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site. An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans. Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used. In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively. In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences. This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion. In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome. The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain. The mechanistic implications and evolutionary relevance of these findings are discussed.

Gene organization in the multiple DNA inversion region min of plasmid p15B of E.coli 15T-: assemblage of a variable gene.

The bacteriophage P1-related plasmid p15B of E. coli 15T- contains a 3.5 kb long region which frequently undergoes complex rearrangements by DNA inversion. Site-specific recombination mediated by the Min DNA invertase occurs at six crossover sites and it eventually results in a population of 240 isomeric configurations of this region. We have determined 8.3-kb sequences of the invertible DNA and its flanking regions. The result explains how DNA inversion fuses variable 3' parts to a constant 5' part, thereby alternatively assembling one out of six different open reading frames (ORF). The resulting variable gene has a coding capacity of between 739 and 762 amino acids. A large portion of its constant part is composed of repeated sequences. The p15B sequences in front of the variable fusion gene encode a small ORF and a phage-specific late promoter and are highly homologous to P1 DNA. Adjacent to the DNA invertase gene min, we have found a truncated 5' region of a DNA invertase gene termed psi cin which is highly homologous to the phage P1 cin gene. Its recombinational enhancer segment is inactive, but it can be activated by the substitution of two nucleotides.

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.

Site-specific DNA recombination system Min of plasmid p15B: a cluster of overlapping invertible DNA segments.

Plasmid p15B of Escherichia coli 15T- carries a 3.5-kilobase segment that undergoes frequent DNA inversion mediated by the DNA inversion enzyme Min, a member of the Din family of site-specific recombinases. While the previously described Din inversion systems invert a DNA segment between two crossover sites in inverted orientation, the Min system produces more complex DNA rearrangements. These have been physically characterized by electron microscopy and by restriction cleavage analysis. The results can best be explained by a model that involves six crossover sites (called mix) and predicts 240 isomeric forms of the invertible region. The model was confirmed by sequencing the six mix sites in plasmids that contain the invertible DNA segments in a frozen configuration. All mix sites fit the dix consensus sequence, and they are all good substrates for DNA inversion when carried in inverted orientation. Recombination between two mix sites in direct orientation was rare, in line with the notion that Din inversion systems are topologically biased to the inversion reaction. Another recently described multiple inversion system, the shufflon of the E. coli plasmid R64, is neither functionally nor structurally related to the Min system of p15B.

Role of the central dinucleotide at the crossover sites for the selection of quasi sites in DNA inversion mediated by the site-specific Cin recombinase of phage P1.

The crossover sites for Cin-mediated inversion consist of imperfect 12 bp inverted repeats with non-palindromic dinucleotides at the center of symmetry. Inversion is believed to occur in vivo between the homologous central 2 bp crossover sequences at the inversely repeated crossover sites through introduction of 2 bp staggered cuts and subsequent reciprocal strand exchanges. The site-specific Cin recombinase acts not only on the normal crossover sites but also, less efficiently, on quasi crossover sites which have some homology with the normal sites. We identified 15 new quasi sites including 4 sites within the cin structural gene. Homology at the 2 bp crossover sequences between recombining sites favors selection as quasi crossover sites. The Cin enzyme can occasionally mediate inversion between nonidentical crossover sequences and such recombinations often result in localized mutations including base pair substitutions and deletions within the 2 bp crossover sequences. These mutations are explained as the consequences of heteroduplex molecules formed between the staggered dinucleotides and either their subsequent resolution by DNA replication or subsequent mismatch repair. Occasional utilization of quasi crossover sites and localized mutagenesis at the crossover sequences in enzyme-mediated inversion processes would be one of the mechanisms contributing to genetic diversity.

Localized conversion at the crossover sequences in the site-specific DNA inversion system of bacteriophage P1.

The crossover sites for site-specific C inversion consist of imperfect 12 bp inverted repeats with the dinucleotide TT at the center of symmetry. The phage P1 Cin recombinase acts not only at these cix sites but also less efficiently at cix-related sequences called quasi-cix sites, cixQ. When cixQ contains a central dinucleotide TT, crossover occurs in vivo at the 2 bp sequence TT in the normal and the quasi-cix sites. If cixQ carries only one T residue, inversion-associated localized conversion can occur at the mismatched position within the 2 bp sequence. The results indicate that Cin generates 2 bp staggered cuts in vivo and that reciprocal strand exchanges occur at these 2 bp crossover sequences.

Insertion element IS1 can generate a 10-base pair target duplication.

Transposable element IS1 is known to generate mainly 9-bp and occasionally 8-bp target duplications upon transposition. We have isolated a plasmid pBR322 derivative having IS1 inserted into a site between the promoter and the structural gene for tetracycline resistance. DNA sequence analysis revealed that integration of this IS1 resulted in a 10-bp target duplication.

Terminal inverted repeats of prokaryotic transposable element IS186 which can generate duplications of variable length at an identical target sequence.

The insertion element IS186, which resides in the chromosome of Escherichia coli K-12, is 1338 bp long. Its termini represent 23-bp perfectly inverted repeats, but a variant carries a mismatch at position 23. IS186 transposes preferentially into G + C-rich sequences and generates target duplications of variable length, even at the same integration site.

Crossover sites cix for inversion of the invertible DNA segment C on the bacteriophage P7 genome.

The bacteriophage P7 genome contains an invertible DNA segment called C which determines its host range. P7 C(+) phages produce plaques on Escherichia coli K12. The C segment consists of a 3-kb unique sequence and 0.62-kb inverted repeats of which one carries an internal 0.2-kb deletion. This deletion has been mapped within the right inverted repeat in the C(+) orientation. The crossover sites cix for inversion of the C segment do not map at the inside boundaries of the inverted repeats, as had been proposed. They are localized at the external ends of these repeats. Thus organization of the C segment in phage P7 is analogous to that in the related phage P1.

Transposable element IS1 intrinsically generates target duplications of variable length.

Target duplication during transposition is one of the characteristics of mobile genetic elements. IS1, a resident insertion element of Escherichia coli K-12, was known to generate a 9-base-pair target duplication, while an IS1 variant, characterized by a nucleotide substitution in one of its terminal inverted repeats, was reported to duplicate 8 base pairs of its target during cointegration. We have constructed a series of transposons flanked by copies of either the normal or the variant IS1. The analysis of their transposition products revealed that transposons with normal termini as well as those with variant termini can intrinsically generate either 9- or 8-base-pair target duplications. We also observed that a normal IS1 from the host chromosome generated an 8-base-pair repeat. The possible relevance of the observation for the understanding of transposition processes and models to explain the variable length of target duplications are discussed.

Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12.

The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671. Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671. However, a rare Bla+ revertant which still retains all the previously acquired drug resistance markers was isolated. On this revertant, the 5' part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored. In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted. Despite of this rearrangement, the split 3' part of bla and its adjacent sequences have remained unchanged. Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed.

Genome fusion mediated by the site specific DNA inversion system of bacteriophage P1.

The genome of bacteriophage P1 contains a segment which is invertible by site specific recombination between sequences near the outside ends of the inverted repeats which flank it. Immediately adjacent to this C segment is the coding sequence for cin, the enzyme catalyzing inversion. We show that multicopy plasmids carrying cin and the sequences at which it acts (cix) can form dimers in the absence of the host recA function. Further, such plasmids can be cotransduced with P1 markers at high frequency from recA lysogens, indicating cointegration with the P1 genome. It is thus demonstrated that a system whose primary role is the inversion of a specific DNA segment can also mediate intermolecular recombination.

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.