Genetic study of the effect of lambda phage on Mu phage production in Escherichia coli K-12 strains with Mu--lambda--Mu structures

The interaction of temperate bacteriophages Mu and lambda is studied during their simultaneous induction in specially constructed heterolysogenic strains of Escherichia coli bearing trimeric Mu--lambda--Mu structures. These strains were obtained by the MU-mediated integration of phage lambda circular genomes. Heterolysogenic strains of E. coli were used for studying phage lambda eliminating effect on Mu development with a simultaneous induction of prophages in the same cell. The results of the study allow the localization of the region of phage lambda genome incorporating gene (genes) lambda, which produces an eliminating effect on Mu development.     

Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.     

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.     

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.     

Fate of plasmids containing Mu DNA: chromosome association and mobilization

Summary The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially all of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase-and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.     

Two-dimensional display of lambda and Escherichia coli restriction fragments separated by Hg-Cs2SO4 centrifugation and gel electrophoresis

EcoRI restriction fragments derived from the DNA of bacteriophage lambda and Escherichia coli were fractionated by density gradient centrifugation of their mercury complexes in Cs2SO4 and subsequent electrophoresis on a horizontal agarose-gel slab. In this two-dimensional display, lambda fragments were resolved into six components and E coli fragments into more than 108 components. Bacterial chromosome regions contiguous to lambda prophage integrated at different sites were amplified by induction, and the EcoRI fragments were subjected to the two-dimensional analysis. As expected, the sets of amplified fragments were clearly different among the various lysogens. The approximate genome region affected by induction was estimated as one-tenth of the whole chromosome.     

Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

In Vivo Cloning of Arylsulfatase-Tyramine Oxidase Genes in rec Strains of Klebsiella aerogenes

A general, genetic technique for in vivo cloning of bacterial genes is presented. We previously introduced the Mu phage into various genera of bacteria including Klebsiella aerogenes with RP4 : : Mu. Using these strains carrying RP4 : : Mu cts and thermo-inducible Mu prophage in the chromosome, we cloned in vivo the arylsulfatase (ats) and tyramine oxidase (tyn) genes by partial thermo-induction. The donor strains carrying the recombinant plasmids were conjugated with K. aerogenes rec strains, which were isolated as UV-sensitive mutants. The resultant recombinant plasmids, pAT1 and pAT2, were purified and used for the transformation of mutant strains deficient in the ats and tyn genes. The ats-tyn genes seemed to be transposed into the RP4::Mu cts plasmid together with other chromosomal DNA fragments. This in vivo cloning method is applicable to a wide variety of gram-negative bacteria.     

Internal EcoRI-generated deletion in prophage Mu DNA

The hybrid plasmid consisting of the plasmid pRP1.2 (derivative of RP4) genome and deleted prophage Mucts 62 genome which lost the central EcoRI fragment of DNA was constructed. The ability of deleted Mu phage to carry out E. coli chromosomal genes transposition was still retained.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Stimulation of mutations suppressing the loss of replication control by small alcohols

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.     

Events following prophage Mu induction.

Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.     

Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112

D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.