Mismatch repair and recombination in E. coli

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

Disruption of T-even Bacteriophages by Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) disrupted T-even bacteriophages as well as lambda bacteriophage. The component substructures of T2L, T4B01, or T6, in particular heads, were readily isolated after treatment with 67% DMSO (v/v). In contrast, concentrations of DMSO above 50% not only separated heads from tails of bacteriophage lambda but led to degradation of the lambda heads. Examination of the isolated free heads of T-even bacteriophage indicated that a distinct neck substructure was attached to one apex of the head. On some free tails a similar neck substructure was also found at the proximal end of the sheath. The dimensions of this neck substructure were found to be about 130 by 180 A; by virtue of its size and morphological attachment to the free heads, it was concluded that this was a distinct substructure and not an extension of the tail tube.     

High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.     

A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones

An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.     

The interaction of emetine with DNA and its effect on the adsorption of certain bacteriophages

The interaction of emetine with DNA in solution at low ionic strength did not bring about a detectable change of the spectrum of either DNA or emetine in the ultraviolet region but resulted in stabilization of the secondary structure of DNA. The irreversible interaction of emetine with DNA or bacteriophage coat, which would result in a loss of infectivity of the lambda cI 857, T7 or T4 BOI bacteriophages did not take place. However, emetine decreased the adsorption of the bacteriophage lambda cI 857 onEscherichia coli C 600, probably due to interaction with cellular receptors.     

Cloning fragments of bacteriophage T5 DNA, determining expression of phage-dependent ligase

Cloning vèctor lambda gt-p MB9 has been used for cloning of DNA fragments of bacteriophage T5 produced by EcoR*I activity. One clone contains a DNA fragment of 2.2 Md which has been mapped at 67-71% on the physical map of the genome. Functional studies have shown that bacteriophage lambda gt-T5 can grow on E. coli lights 7. Infection of this E.coli strain with phage lambda gt-T5 induces DNA-ligase activity which has been previously observed in E. Coli infected with bacteriophage T5.     

The purification and properties of the scaffolding protein of bacteriophage lambda.

The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head. Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein. The Nu3 gene has been cloned and overexpressed, and its protein product has been purified. The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction. The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396. A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape.     

Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS)

Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration. IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man). Truncation of IIC(Man) renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).     

A new filamentous phage cloning vector: fd-tet

We have constructed a hybrid chromosome composed of the genome of wild-type fd (a filamentous, male-specific bacteriophage) and a segment of transposon Tn10 coding for tetracycline resistance but not including the Tn10 insertion sequences. The hybrid phage infects male E. coli, thereby transducing the infected cells to tetracycline resistance. The phage DNA can also be propagated in F- cells after transfection. This new phage, fd-tet, may be used as a cloning vector to produce large quantities of cloned DNA in single-stranded form. Its usefulness has been demonstrated by cloning of a fragment from bacteriophage lambda. Some unexpected sequence alterations have been identified in lambda cloning experiments.     

Studies on the arrangement of DNA inside viruses using a breakable bis-psoralen crosslinker

We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40. Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments. In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model. These results imply that the structures of DNA inside these viruses are either highly disordered or very complex.     

Energy-dependent degradation of lambda O protein in Escherichia coli.

Protein O of bacteriophage lambda is a short-lived protein which has a key role in the replication of the phage DNA in Escherichia coli. Here we present evidence that lambda O degradation is energy dependent: it is impaired by cyanide and alpha-methylglucoside, both of which inhibit cellular energy metabolism. Removal of these inhibitors restored the degradation of lambda O. Our experiments suggest that limited amounts of cellular energy are sufficient to support lambda O degradation. In addition, degradation of lambda O protein is prevented by a mutation in the E. coli clpP gene, but not by a mutation in the clpA gene. These results suggest that the ClpP protease is involved in the energy-dependent degradation of the lambda O protein.     

A role for recombination in the production of "free-loader" lambda bacteriophage particles

Density-labeled crosses were performed with bacteriophage lambda under conditions which diminish DNA duplication. The production of viable phage containing fully conserved parental DNA was found to be dependent upon the action of the genetic recombination systems. The production of phage containing DNA with one newly synthesized chain was less dependent upon recombination. The production of phage with chromosomes both of whose chains were synthesized following infection show little, if any, dependence on recombination. One can speculate that some step in the maturation process of bacteriophage lambda is inseparable from the reduction of lambda DNA to the monomeric rods characteristic of lambda virions.     

The effect of the phage lambda ral gene on the level of synthesis of the EcoK restriction endonuclease beta-subunit

E. coli hsd genes were subcloned from lambda 642 (ral+) into lambda L47.1 vector (ral-after replacement). The influence of bacteriophage lambda ral gene on the expression efficiency of hsdS kappa, hsdM kappa genes was investigated. It was shown, that its presence in vitro enhanced the synthesis of beta-subunit, hsdM gene product, and the increase of modification in vivo was observed. It is proposed that the increase of modification rate of lambda phage fully unmodified DNA is connected with the appearance of E. coli DNA methylase consisting of beta- and gamma-subunits but lacking alpha-subunit.     

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.     

Sequence of amino acids in lamB responsible for spontaneous ejection of bacteriophage lambda DNA

The DNA sequence of the promoter-distal half of lamB from Shigella sonnei 3070 has been determined and compared with the known sequence for the Escherichia coli K12 gene. The only predicted amino acid changes in this region of LamB, the receptor protein for bacteriophage lambda, lie between positions 381 and 390, where seven of the ten amino acids are altered. Evidence is presented that indicates that this region is responsible for the ability of the S. sonnei receptor, but not the E. coli receptor, to trigger spontaneous ejection of DNA from the bacteriophage in vitro. DNA injection in vivo must be more complex and involve also the host Pel protein and the lambda tail proteins gpJ, gpH, and gpV.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

IS Elements in Bacteriophage Lambda Gene cI Prevent Expression of Gene rex

IS5 inserted into gene cI of bacteriophage lambda greatly reduces the synthesis of Rex protein. Two other IS insertions in cI are even more polar, but two amber mutations near the 5' end of cI allow normal expression of Rex.