Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases.

Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.     

Effect of the lambda S gene product on properties of the Escherichia coli inner membrane

The S gene of bacteriophage lambda is a late gene required for cell lysis, but unlike the other two lysis genes, R and Rz, it does not code for an endolysin. Earlier studies have shown that the S gene product inhibits respiration and macromolecular synthesis and makes the inner membrane permeable to sucrose. In this study, the effect of the S gene product on a number of Escherichia coli membrane functions (active transport, permeability, respiration, and transhydrogenase and ATPase activity) were measured, and a product of the lambda S gene was identified in the inner membrane fraction by two-dimensional polyacrylamide gel electrophoresis. The results of these experiments indicate that the lambda S product is present in the inner membrane, that it increased the permeability of the membrane for all of the small molecules that were tested, and that its action is reversible. The simplest explanation of these results is that the S gene product forms a hydrophilic pore through the inner membrane, allowing small molecules and lambda lysozyme to pass through.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.     

Transduction of nitrate reductase loci of Escherichia coli by phages P-1 and lambda

Summary A lactate-nitrate medium suitable for genetic studies with nitrate reductase mutants (nar ^-) of Escherichia coli was devised. This permitted the selection of nar ^- strains by their failure to use nitrate as terminal electron acceptor during anaerobic growth, in addition to the selection procedure based on the chlorate resistance of nar ^- mutants. Transduction studies with phage P1 and nar ^- mutants from both sources demonstrated the existence of at least three nar genes in the gal region of the E. coli linkage map, their relative positions being: gal .... narF .... bio .... narD .... narE. Using phage cotransduction of narD with bio was observed and several independently-isolated defective -transducing phages were examined. Phage also transduced the narF gene with gal linkage but the narE gene was not -transducible.     

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Summary Specialized transducing phages asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli, The clockwise order of genes on the chromosome is found to be: bglB, (pst, glmS), (uncA, uncB), criC, asn, trkD, rbs, rrnC, ilv.Abbreviations CCC covalently closed circular - MD 10⁶ Daltons; mw, molecular weight - R. restriction endonuclease - LFT, HFT low (high) frequency transducing - a.p.i. average phage input - m.o.i. multiplicity of infection     

Differential induction of Escherichia coli autolysis by penicillin and the bacteriophage phi X174 gene E product.

The behavior of the temperature-sensitive, penicillin-tolerant Escherichia coli mutant VC44 to endogenously induced autolysis by the bacteriophage phi X174 gene E product (gpE) was investigated. Expression of the cloned phi X174 lysis gene showed that cultures of strain VC44 grown at the restricted temperature were fully sensitive to endogenously induced autolysis. The results revealed that the modes of E. coli lysis induction by gpE and by penicillin differ and that the trigger mechanisms for autolysis depend greatly on the specific inducer used.     

Isolation and characterization of specialized transducing lambda phages carrying ribosomal protein genes of Escherichia coli

Summary Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54°C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.     

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.     

Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.     

Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli.

A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.     

Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein.

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.     

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.     

The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli

The dnaZ protein has been purified to near-homogeneity using an in vitro complementation assay that measures the restoration of activity in a crude enzyme fraction from the dnaZ mutant deficient in the replication of phi X174 DNA. Over 70-fold overproduction of the protein was obtained with a bacteriophage lambda lysogen carrying the dnaZ gene. The purified protein, under reducing and denaturing conditions, has a molecular weight of 52,000 and appears to be a dimer in its native form. The dnaZ protein is judged to be th 52,000-dalton gamma subunit of DNA polymerase III holoenzyme (McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484) for the following reasons: (i) highly purified DNA polymerase III holoenzyme contains a 52,000-dalton polypeptide and has dnaZ-complementing activity; (ii) the 52,000-dalton polypeptide is associated tightly with the DNA polymerase III holoenzyme and can be separated from the DNA polymerase III core only with severe measures; (iii) no other purified replication protein, among 14 tested, contains dnaZ protein activity; and (iv) the abundance of dnaZ protein, estimated at about 10 dimer molecules per Escherichia coli cell, is similar to that of the DNA polymerase III core. Among several circular templates tested in vitro (i.e. single stranded phi X174, G4 and M13 DNAs, and duplex phi X174 DNA), all rely on dnaZ protein for elongation by DNA polymerase III holoenzyme. The protein acts catalytically at a stoichiometry of one dimer per template.