Shigeru Iida

The DNA sequence of an IS1-flanked transposon coding for resistance to chloramphenicol and fusidic acid

(ii) The transposon appeared too small to code for the chloramphenicol resistance gene product (22-24 X lo3 mol. wt [4]), a protein for fusidic acid resistance and a putative ‘transposase’ [5] which would be involved in transposition. Since the expression of chloramphenicol resistance in Enterobacteriaceae is under the control of catabolite repression [6], the sequence of the non-coding regions of the DNA should contain a catabolite repressor binding site (CAP site) which could be compared with the 3 published CAP sites controlling the lac, gal and ara operons of Escherichia coli [7-91. The results indicate that: (a) A gene for a putative ‘transposase’ does not exist; (b) The gene(s) for resistance to chloramphenicol and fusidic acid are either identical or overlap in the same reading frame; (c) A probable CAP site was found N 120 basepairs preceding the initiation of transcription.

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

SummaryE. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cmr lysogens. Lysates of P1CmSmSu lysogens transduced CmrSmrSur at high frequency and they formed plaques with an efficiency of 10-4 to 10-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. CmrSmrSur transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, CmrSmrSur transductants isolated after infection at low multiplicity appeared to carry the CmrSmrSur markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA- condition. The mechanism of formation of plaque forming P1Cm is discussed.

DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies.

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.

Does the insertion element IS1 transpose preferentially into A+T-rich DNA segments?

SummaryIS1-mediated insertion and deletion formation occur preferentially into A+T-rich regions of DNA of bacteriophage P1 and of the r-determinant of the R plasmid NR1. The significance of this correlation is discussed in view of other published data.

Bacteriophage P1 carries two related sets of genes determining its host range in the invertible c segment of its genome

The bacteriophage P1 genome carries an invertible C segment consisting of 3-kb unique sequences flanked by 0.6-kb inverted repeats. Host range mutations of P1 have been mapped in the C segment region. P1 derivatives carrying insertions and deletions in the left half of the C segment in one of two orientations termed C(+) do not affect the plaque-forming ability on Escherichia coli K12 and E coli C, whereas those having insertions in the right half of the C segment fail to form plaques on these hosts. An E. coli C mutant which allows the latter insertion mutants with the C segment in the C(-) configuration to form plaques has been isolated. Not only P1 C(-) but also P1 C(+) phages gave plaques on this E. coli C mutant. The results are consistent with the notion that the C segment of P1 carries two sets of genes for host specificity, and that C inversion alters the P1 host range through activation of one set of the genes. Furthermore, extended host range mutants can be isolated by point mutation in either set of the P1 genes. C inversion is a slow process, but it occurs on the phage genome upon its vegetative growth as well as on the prophage in the lysogenic state. The 3-kb invertible G segment of the phage Mu genome is known to be homologous with the central 3-kb part of the C segment of P1 and to carry also two sets of genes for Mu host specificity. While only Mu G(-) grows on E. coli C, both Mu G(+) and Mu G(-) phages form plaques on the E. coli C mutant sensitive to P1 C(-). In the discussion the gene organization of the P1 C segment is compared with that of the Mu G segment.

Two DNA antirestriction systems of bacteriophage P1, darA, and darB: characterization of darA− phages

Bacteriophage P1 is only weakly restricted when it infects cells carrying type I restriction and modification systems even though DNA purified from P1 phage particles is a good substrate for type I restriction enzymes in vitro. Here we show that this protection against restriction is due to the products of two phage genes which we call darA and darB (dar for defense against restriction). Each of the dar gene products provides protection against a different subset of type I restriction systems. The darA and darB gene products are found in the phage head and protect any DNA packaged into a phage head, including transduced chromosomal markers, from restriction. The proteins must, therefore, be injected into recipient cells along with the DNA. The proteins act strictly in cis. For example, upon double infection of restricting cells with dar+ and dar- P1 phages, the dar+ genomes are protected from restriction while the dar- genomes are efficiently restricted.

Functional characterization of the prokaryotic mobile genetic element IS26

SummaryIS26L and IS26R are the 820 bp long elements found as direct repeats at both ends of the kanamycin resistance transposon Tn2680. They can mediate cointegration in E. coli K12 which contains no IS26 in its chromosome. Cointegration occurs in rec+ or recA- strains with similar frequency. Upon cointegration mediated by either IS26R or IS26L, the element is duplicated and integrated into one of many different sites. Both IS26L and IS26R carry 14 bp perfect terminal inverted repeats and generate 8 bp direct repeats at their target sequences. Deletion formation mediated by IS26R was also observed. These functional and structural features of IS26 are characteristic of a prokaryotic mobile genetic element.

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.

Gene organization and target specificity of the prokaryotic mobile genetic element IS26

SummaryThe 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.

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

SummaryThe crossover sites for Cin-mediated inversion consist of imperfect 12 bp inverted repeats with non-palindomic 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 tubsequent 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.